Binding molecule specific for cd73 and use of binding molecule

ABSTRACT

A binding molecule specific for CD73 and a use of the binding molecule. Specifically, provided are a separate antibody binding CD73 and inhibiting the activity of CD73 or an antigen binding part of the separate antibody, and a use of the separate antibody or the antigen binding part thereof in treatment of diseases; also provided are a nucleic acid molecule encoding the separate antibody or the antigen binding part thereof, an expression vector for expressing the separate antibody or the antigen binding part thereof, a host cell, and a preparation method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.17/240,141, filed Apr. 26, 2021, now U.S. Pat. No. 11,174,319, which isa continuation application of Int'l Appl. No. PCT/CN2020/094489, filedJun. 5, 2020, which claims priority to PCT/CN2019/090366, filed Jun. 6,2019, each and all of which are incorporated herein by reference intheir entirety.

SEQUENCE DISCLOSURE

This application includes as part of its disclosure an electronicsequence listing text file named “1158498o003602.txt”, having a size of196,332 and created Sep. 15, 2021, which is hereby incorporated byreference in its entirety.

TECHNICAL FIELD

This invention relates to the isolated antibody or antigen-bindingfragment thereof specifically binding to CD73, this invention alsorelates to the use of the isolated antibody or antigen-binding fragmentthereof in treatment of diseases, and this invention relates to thetherapeutic method of the isolated antibody or antigen-binding fragmentthereof.

BACKGROUND ART

CD73 (ecto-5′-nucleotidase) is membrane-bound/free protein with a sizeof about 70 kDa and consists of N-terminal and C-terminal subunits and aflexible link. The C-terminus is anchored to the cell membrane throughglycosyl-phosphatidylinositol (GPI). CD73 exerts an enzymatic activitythrough switching between open and closed conformational states (Knapp,K. et al. (2012), Structure, 20(12), 2161-2173). In the adenosinepathway, CD73 can continue to hydrolyze AMP produced by CD39 toadenosine. Adenosine exerts various immunosuppressive effects, such asinhibiting the proliferation of CD4+ T and CD8+ T, inhibiting NK cellactivity, promoting Treg proliferation, and the like.

It has been widely reported that CD73 was highly expressed in varioustumor such as gastric cancer, triple negative breast cancer,non-small-cell adenocarcinoma, rectal adenocarcinoma, colorectal cancer,renal cell carcinoma, ovarian cancer, prostate cancer, oral squamouscell carcinoma, head and neck squamous cell carcinoma and so on, andhigh CD73 expression was associated with poor prognosis (Vijayan, D. etal., (2017), Nature reviews Cancer, 17, 709). Increased expression ofCD73 in tumor microenvironment was associated with tumor proliferation,metastasis, neovascularization, and poor survival of the patients(Allard, B. et al., (2017), Immunological reviews, 276 (1), 121-144).With the high expression of CD73, the increased adenosine inhibits tumorimmunity by activating tumor intrinsic and host-mediated tumor-promotingmechanisms, restricting immune cell infiltration and production ofcytotoxicity and cytokines (such as interferon), and causes strongimmunosuppression (Ohta, A. et al., (2016), Front Immunol, 7, 109).Immunosuppression is a typical characteristic of cancer, and it isimportant to overcome the inhibitory barrier. Therefore, it is a highlypotential therapeutic direction of inhibiting the production ofadenosine in the tumor microenvironment by inhibiting CD73, therebyactivating immunity and inhibiting tumor growth.

Antibodies can inhibit the enzymatic activity of CD73 by antagonizingCD73 allosteric to the active form, but there is no report of anyantibody that can directly bind to the CD73 active site to inhibit itsenzyme activity. Meanwhile, although CD73 is a very potentialtherapeutic target, more research is needed to verify the prognosis ofCD73 targets and to develop related drugs and combination therapies.

SUMMARY OF INVENTION

The invention provide an isolated antibody or antigen-binding fragmentspecifically binding for CD73 and uses thereof in the treatmentdiseases.

In one respect, the invention provide an isolated antibody orantigen-binding fragment thereof comprising a heavy chain variableregion that comprises HCDR1, HCDR2, HCDR3; and a light chain variableregion that comprises LCDR1, LCDR2, LCDR3, wherein:

-   -   (a) HCDR1 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs: 5, 18, 31, 43 and 56, and        conservative modifications thereof;    -   (b) HCDR2 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs: 6, 19, 32, 44 and 57, and        conservative modifications thereof;    -   (c) HCDR3 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs: 7, 20, 33, 45 and 58, and        conservative modifications thereof;    -   (d) LCDR1 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs: 10, 23, 36, 48 and 61, and        conservative modifications thereof;    -   (e) LCDR2 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs: 11, 24, 37, 49 and 62, and        conservative modifications thereof; and    -   (f) LCDR3 comprises an amino acid sequence selected from the        group consisting of SEQ ID NOs: 12, 25, 38, 50 and 63, and        conservative modifications thereof.

In some embodiments, the isolated antibody or antigen-binding fragmentthereof comprises:

-   -   1) (a) HCDR1 comprising SEQ ID NO: 5, (b) HCDR2 comprising SEQ        ID NO: 6, (c) HCDR3 comprising SEQ ID NO: 7, (d) LCDR1        comprising SEQ ID NO: 10, (e) LCDR2 comprising SEQ ID NO: 11,        and (f) LCDR3 comprising SEQ ID NO: 12;    -   2) (a) HCDR1 comprising SEQ ID NO: 18, (b) HCDR2 comprising SEQ        ID NO: 19, (c) HCDR3 comprising SEQ ID NO: 20, (d) LCDR1        comprising SEQ ID NO: 23, (e) LCDR2 comprising SEQ ID NO: 24,        and (f) LCDR3 comprising SEQ ID NO: 25;    -   3) (a) HCDR1 comprising SEQ ID NO: 31, (b) HCDR2 comprising SEQ        ID NO: 32, (c) HCDR3 comprising SEQ ID NO: 33, (d) LCDR1        comprising SEQ ID NO: 36, (e) LCDR2 comprising SEQ ID NO: 37,        and (f) LCDR3 comprising SEQ ID NO: 38;    -   4) (a) HCDR1 comprising SEQ ID NO: 43, (b) HCDR2 comprising SEQ        ID NO: 44, (c) HCDR3 comprising SEQ ID NO: 45, (d) LCDR1        comprising SEQ ID NO: 48, (e) LCDR2 comprising SEQ ID NO: 49,        and (f) LCDR3 comprising SEQ ID NO: 50; or    -   5) (a) HCDR1 comprising SEQ ID NO: 56, (b) HCDR2 comprising SEQ        ID NO: 57, (c) HCDR3 comprising SEQ ID NO: 58, (d) LCDR1        comprising SEQ ID NO: 61, (e) LCDR2 comprising SEQ ID NO: 62,        and (f) LCDR3 comprising SEQ ID NO: 63.

In some embodiments, the isolated antibody or antigen-binding fragmentthereof comprises: (a) HCDR1 comprising SEQ ID NO: 5, (b) HCDR2comprising SEQ ID NO: 6, (c) HCDR3 comprising SEQ ID NO: 7, (d) LCDR1comprising SEQ ID NO: 10, (e) LCDR2 comprising SEQ ID NO: 11, and (f)LCDR3 comprising SEQ ID NO: 12.

In some embodiments, the isolated antibody or antigen-binding fragmentthereof comprises: (a) HCDR1 comprising SEQ ID NO: 18, (b) HCDR2comprising SEQ ID NO: 19, (c) HCDR3 comprising SEQ ID NO: 20, (d) LCDR1comprising SEQ ID NO: 23, (e) LCDR2 comprising SEQ ID NO: 24, and (f)LCDR3 comprising SEQ ID NO: 25.

In some embodiments, the isolated antibody or antigen-binding fragmentthereof comprises: (a) HCDR1 comprising SEQ ID NO: 31, (b) HCDR2comprising SEQ ID NO: 32, (c) HCDR3 comprising SEQ ID NO: 33, (d) LCDR1comprising SEQ ID NO: 36, (e) LCDR2 comprising SEQ ID NO: 37, and (f)LCDR3 comprising SEQ ID NO: 38.

In some embodiments, the isolated antibody or antigen-binding fragmentthereof comprises: (a) HCDR1 comprising SEQ ID NO: 43, (b) HCDR2comprising SEQ ID NO: 44, (c) HCDR3 comprising SEQ ID NO: 45, (d) LCDR1comprising SEQ ID NO: 48, (e) LCDR2 comprising SEQ ID NO: 49, and (f)LCDR3 comprising SEQ ID NO: 50.

In some embodiments, the isolated antibody or antigen-binding fragmentthereof comprises: (a) HCDR1 comprising SEQ ID NO: 56, (b) HCDR2comprising SEQ ID NO: 57, (c) HCDR3 comprising SEQ ID NO: 58, (d) LCDR1comprising SEQ ID NO: 61, (e) LCDR2 comprising SEQ ID NO: 62, and (f)LCDR3 comprising SEQ ID NO: 63.

In some embodiments, the isolated antibody or antigen-binding fragmentthereof comprises:

-   -   (i) the heavy chain variable region (VH) comprising an amino        acid sequence which has at least 85% identity to the amino acid        sequences selected from the group consisting of SEQ ID NOs: 3,        16, 29, 41, 54, 66, 67, 76, 77 and 78, and conservative        modifications thereof; and    -   (ii) the light chain variable region (VL) comprising an amino        acid sequence which has at least 85% identity to the amino acid        sequence selected from the group consisting of SEQ ID NOs: 8,        21, 34, 46, 59, 68, 69, 70, 79 and 80, and conservative        modifications thereof.

In some embodiments, the heavy chain variable regions comprise an aminoacid sequence which has at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identity to the heavyvariable regions selected from the group of (i), and the light chainvariable regions comprise an amino acid sequence which has at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identity to the light variable regions selected from the group of(ii).

In some embodiments, the isolated antibody or antigen-binding fragmentthereof comprises:

-   -   1) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 3, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 8;    -   2) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 16, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identical to the amino acid sequences of SEQ ID NO:21;    -   3) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 29, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 34;    -   4) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 41, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 46;    -   5) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 54, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 59;    -   6) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 66, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 68;    -   7) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 66, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 69;    -   8) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 66, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 70;    -   9) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 67, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 68;    -   10) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 67, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 69;    -   11) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 67, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 70;    -   12) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 76, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 79;    -   13) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 76, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 80;    -   14) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 77, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 79;    -   15) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 77, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 80;    -   16) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 78, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 79;    -   17) a heavy chain variable region (VH) that comprises an amino        acid sequence which has at least 85% identity to the amino acid        sequences of SEQ ID NO: 78, and a light chain variable region        (VL) that comprises an amino acid sequence which has at least        85% identity to the amino acid sequences of SEQ ID NO: 80.

In some embodiments, the isolated antibody or antigen-binding fragmentthereof, wherein the heavy and light chain variable regions comprise anamino acid sequence at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identity to the heavy and lightchain variable regions, respectively, selected from the group consistingof 1)-17).

In some embodiments, the isolated antibody is an IgG.

In some embodiments, the isolated antibody is an IgG1, IgG2 or IgG4.

In some embodiments, the isolated antibody is a monoclonal antibody, achimeric antibody, a humanized antibody, a human engineered antibody, ahuman antibody, Fv, a single chain antibody (scFv), Fab, Fab′, Fab′-SHor F(ab′)₂.

In some embodiments, the isolated antibody or antigen-binding fragmentthereof comprising a heavy chain and a light chain, wherein:

-   -   (I) the heavy chain comprising an amino acid sequence which is        at least 85% identity to the amino acid sequences selected from        the group consisting of SEQ ID NOs: 13, 15, 26, 27, 39, 51, 52,        64, 71, 72, 81, 82, 83, 86, 87, 90, 91, 124, 125, 126 and 127,        and conservative modifications thereof; and    -   (II) the light chain comprising an amino acid sequence which is        at least 85% identity to the amino acid sequences selected from        the group consisting of SEQ ID NOs: 14, 28, 40, 53, 65, 73, 74,        75, 84, 85, 88 and 89, and conservative modifications thereof.

In some embodiments, the heavy chain comprises an amino acid sequencewhich has at least 90%, at least 95%, at least 96%, at least 97%,atleast 98%, or 100% identity to the heavy chains selected from the groupof (I), and the light chain comprises an amino acid sequence which hasat least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or100% identity to the light chain selected from the group of (II).

In some embodiments, the isolated antibody or antigen-binding fragmentthereof comprises:

-   -   1) a heavy chain that comprises an amino acid sequence which is        at least 85% identity to the amino acid sequences of SEQ ID NO:        13, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 14;    -   2) a heavy chain that comprises an amino acid sequence which has        at least 85% identity to the amino acid sequences of SEQ ID NO:        15, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 14;    -   3) a heavy chain that comprises an amino acid sequence which has        at least 85% identity to the amino acid sequences of SEQ ID NO:        26, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 28;    -   4) a heavy chain that comprises an amino acid sequence which has        at least 85% identity to the amino acid sequences of SEQ ID NO:        27, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 28;    -   5) a heavy chain that comprises an amino acid sequence which has        at least 85% identity to the amino acid sequences of SEQ ID NO:        39, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 40;    -   6) a heavy chain that comprises an amino acid sequence which has        at least 85% identity to the amino acid sequences of SEQ ID NO:        51, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 53;    -   7) a heavy chain that comprises an amino acid sequence which has        at least 85% identity to the amino acid sequences of SEQ ID NO:        52, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 53;    -   8) a heavy chain that comprises an amino acid sequence which has        at least 85% identity to the amino acid sequences of SEQ ID NO:        64, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 65;    -   9) a heavy chain that comprises an amino acid sequence which has        at least 85% identity to the amino acid sequences of SEQ ID NO:        71, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 73;    -   10) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 71, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 74;    -   11) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 71, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 75;    -   12) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 72, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 73;    -   13) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 72, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 74;    -   14) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 72, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 75;    -   15) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 81, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 84;    -   16) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 81, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 85;    -   17) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 82, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 84;    -   18) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 82, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 85;    -   19) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 83, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 84;    -   20) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 83, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 85;    -   21) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 90, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 84;    -   22) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 90, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 85;    -   23) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 91, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 84;    -   24) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 91, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 85;    -   25) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 86, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 88;    -   26) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 86, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 89;    -   27) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 87, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 88;    -   28) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 87, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 89;    -   29) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 124, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 84;    -   30) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 124, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 85;    -   31) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 125, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 84; or    -   32) a heavy chain that comprises an amino acid sequence which        has at least 85% identity to the amino acid sequences of SEQ ID        NO: 125, and a light chain that comprises an amino acid sequence        which has at least 85% identity to the amino acid sequence of        SEQ ID NO: 85.

In some embodiments, the heavy and light chain comprise an amino acidsequence at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identity to the heavy and light chainvariable regions, respectively, selected from the group consisting of1)-32).

In another respect, the invention provides an isolated antibody orantigen-binding fragment thereof, which comprises a heavy chain of SEQID NO: 71 and a light chain of SEQ ID NO: 73.

In yet another respect, the invention provides an isolated antibody orantigen-binding fragment thereof, which comprises a heavy chainconsisting essentially of SEQ ID NO: 72 and a light chain consistingessentially of SEQ ID NO: 74.

In one respect, the invention provides an isolated antibody orantigen-binding fragment thereof, which comprises a heavy chain of SEQID NO: 72 and a light chain of SEQ ID NO: 75.

In another respect, the invention provides an isolated antibody orantigen-binding fragment thereof, which comprises a heavy chainconsisting essentially of SEQ ID NO: 81 and a light chain consistingessentially of SEQ ID NO: 85.

In yet another respect, the invention provides an isolated antibody orantigen-binding fragment thereof, which comprises a heavy chainconsisting essentially of SEQ ID NO: 82 and a light chain consistingessentially of SEQ ID NO: 84.

In one respect, the invention provides an isolated antibody orantigen-binding fragment thereof, which comprises a heavy chainconsisting essentially of SEQ ID NO: 83 and a light chain consistingessentially of SEQ ID NO: 85.

In another respect, the invention provides an isolated antibody orantigen-binding fragment thereof, which comprises a heavy chainconsisting essentially of SEQ ID NO: 124 and a light chain consistingessentially of SEQ ID NO: 85.

In yet another respect, the invention provides an isolated antibody orantigen-binding fragment thereof, which comprises a heavy chainconsisting essentially of SEQ ID NO: 125 and a light chain consistingessentially of SEQ ID NO: 85.

In some embodiments, the isolated antibody or antigen-binding fragmentthereof is an antagonist of CD73 or 5′-nucleotidase of CD73.

In some embodiments, the CD73 is human CD73.

In one respect, the invention provides a method of decreasing adenosinelevels in a subject with tumor, comprising administering atherapeutically effective amount of the isolated antibody orantigen-binding fragment thereof.

In another respect, the invention provides a method of improving a Tcell response in a subject with tumor, comprising administering atherapeutically effective amount of the isolated antibody orantigen-binding fragment thereof.

In yet another respect, the invention provides a method of stimulatingan immune response in a subject comprising administering atherapeutically effective amount of the isolated antibody orantigen-binding fragment thereof.

In some embodiments, the subject is the subject with tumor.

In some embodiments, the subject has a tumor cell expressing CD73 and/ora tumor microenvironment containing CD73.

In one respect, the invention provides a method for inhibiting thegrowth of tumor cells in a subject comprising administering to thesubject a therapeutically effective amount of the isolated antibody orantigen-binding fragment thereof.

In another respect, the invention provides use of the isolated antibodyor antigen-binding fragment thereof in the manufacture of a medicamentfor decreasing adenosine levels in a tumor cell and/or a tumormicroenvironment.

In yet another respect, the invention provides use of the isolatedantibody or antigen-binding fragment thereof in the manufacture of amedicament for stimulating a T cell response in a subject with tumor.

In one respect, the invention provides use of the isolated antibody orantigen-binding fragment thereof in the manufacture of a medicament forstimulating an immune response in a subject.

In some embodiments, the subject is a subject with tumor.

In some embodiments, the subject has a tumor cell expressing CD73 and/ora tumor microenvironment containing CD73.

In another respect, the invention provides use of the isolated antibodyor antigen-binding fragment thereof in the manufacture of a medicamentfor inhibiting the growth of tumor cells in a subject.

In yet another respect, the invention provides the isolated antibody orantigen-binding fragment thereof for use in decreasing adenosine levelsin a tumor cell and/or a tumor microenvironment.

In one respect, the invention provides the isolated antibody orantigen-binding fragment thereof for use in stimulating a T cellresponse in a subject with tumor.

In another respect, the invention provides the isolated antibody orantigen-binding fragment thereof for use in stimulating an immuneresponse in a subject.

In some embodiments, the subject is a subject with tumor.

In some embodiments, the subject has a tumor cell expressing CD73 and/ora tumor microenvironment containing CD73.

In another respect, the invention provides the isolated antibody orantigen-binding fragment thereof for use in inhibiting the growth oftumor cells in a subject.

In yet another respect, the invention provides an isolated nucleic acidcomposition, which comprises:

-   -   (I) a first nucleic acid comprising a nucleotide sequence        selected from the group consisting of SEQ ID NOs: 4, 17, 30, 42,        55, 92, 93, 102, 103 and 104; and    -   (II) a second nucleic acid comprising a nucleotide sequence        selected from the group consisting of SEQ ID NOs: 9, 22, 35, 47,        60, 94, 95, 96, 105 and 106.

In some embodiments, the isolated nucleic acid composition comprises:

-   -   1) the first nucleic acid comprising SEQ ID NO: 4 and the second        nucleic acid comprising SEQ ID NO: 9;    -   2) the first nucleic acid comprising SEQ ID NO: 17 and the        second nucleic acid comprising SEQ ID NO: 22;    -   3) the first nucleic acid comprising SEQ ID NO: 30 and the        second nucleic acid comprising SEQ ID NO: 35;    -   4) the first nucleic acid comprising SEQ ID NO: 42 and the        second nucleic acid comprising SEQ ID NO: 47;    -   5) the first nucleic acid comprising SEQ ID NO: 55 and the        second nucleic acid comprising SEQ ID NO: 60;    -   6) the first nucleic acid comprising SEQ ID NO: 92 and the        second nucleic acid comprising SEQ ID NO: 94;    -   7) the first nucleic acid comprising SEQ ID NO: 92 and the        second nucleic acid comprising SEQ ID NO: 95;    -   8) the first nucleic acid comprising SEQ ID NO: 92 and the        second nucleic acid comprising SEQ ID NO: 96;    -   9) the first nucleic acid comprising SEQ ID NO: 93 and the        second nucleic acid comprising SEQ ID NO: 94;    -   10) the first nucleic acid comprising SEQ ID NO: 93 and the        second nucleic acid comprising SEQ ID NO: 95;    -   11) the first nucleic acid comprising SEQ ID NO: 93 and the        second nucleic acid comprising SEQ ID NO: 96;    -   12) the first nucleic acid comprising SEQ ID NO: 102 and the        second nucleic acid comprising SEQ ID NO: 105;    -   13) the first nucleic acid comprising SEQ ID NO: 102 and the        second nucleic acid comprising SEQ ID NO: 106;    -   14) the first nucleic acid comprising SEQ ID NO: 103 and the        second nucleic acid comprising SEQ ID NO: 105;    -   15) the first nucleic acid comprising SEQ ID NO: 103 and the        second nucleic acid comprising SEQ ID NO: 106;    -   16) the first nucleic acid comprising SEQ ID NO: 104 and the        second nucleic acid comprising SEQ ID NO: 105; or    -   17) the first nucleic acid comprising SEQ ID NO: 104 and the        second nucleic acid comprising SEQ ID NO: 106.

In one respect, the invention provides an expression vector composition,which comprises:

-   -   (I) a first expression vector comprising a nucleotide sequence        selected from the group consisting of SEQ ID NOs: 4, 17, 30, 42,        55, 92, 93, 102, 103 and 104; and    -   (II) a second expression vector comprising a nucleotide        sequences selected from the group consisting of SEQ ID NOs: 9,        22, 35, 47, 60, 94, 95, 96, 105 and 106.

In some embodiments, the expression vector composition comprises:

-   -   1) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 4 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 9;    -   2) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 17 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 22;    -   3) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 30 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 35;    -   4) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 42 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 47;    -   5) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 55 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 60;    -   6) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 92 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 94;    -   7) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 92 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 95;    -   8) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 92 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 96;    -   9) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 93 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 94;    -   10) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 93 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 95;    -   11) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 93 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 96;    -   12) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 102 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 105;    -   13) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 102 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 106;    -   14) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 103 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 105;    -   15) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 103 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 106;    -   16) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 104 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 105; or    -   17) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 104 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 106.

In another respect, the invention provides an expression vector, whichcomprises:

-   -   (I) a first nucleic acid sequence comprising a nucleotide        sequence selected from the group consisting of SEQ ID NOs: 4,        17, 30, 42, 55, 92, 93, 102, 103 and 104; and    -   (II) a second nucleic acid sequence comprising a nucleotide        sequence selected from the group consisting of SEQ ID NOs: 9,        22, 35, 47, 60, 94, 95, 96, 105 and 106.

In some embodiments, the expression vector, comprises:

-   -   1) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 4 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 9;    -   2) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 17 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 22;    -   3) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 30 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 35;    -   4) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 42 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 47;    -   5) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 55 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 60;    -   6) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 92 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 94;    -   7) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 92 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 95;    -   8) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 92 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 96;    -   9) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 93 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 94;    -   10) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 93 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 95;    -   11) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 93 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 96;    -   12) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 102 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 105;    -   13) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 102 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 106;    -   14) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 103 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 105;    -   15) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 103 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 106;    -   16) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 104 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 105;    -   17) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 104 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 106.

In yet another respect, the invention provides an isolated nucleic acidcomposition, which comprises:

-   -   (I) a first nucleic acid comprising a nucleotide sequence        selected from the group consisting of SEQ ID NOs: 97, 98, 107,        108, 109, 112, 113, 116 and 117; and    -   (II) a second nucleic acid comprising a nucleotide sequence        selected from the group consisting of SEQ ID NOs: 99, 100, 101,        110, 111, 114 and 115.

In some embodiments, the isolated nucleic acid composition, comprises:

-   -   1) the first nucleic acid comprising SEQ ID NO: 97 and the        second nucleic acid comprising SEQ ID NO: 99;    -   2) the first nucleic acid comprising SEQ ID NO: 97 and the        second nucleic acid comprising SEQ ID NO: 100;    -   3) the first nucleic acid comprising SEQ ID NO: 97 and the        second nucleic acid comprising SEQ ID NO: 101;    -   4) the first nucleic acid comprising SEQ ID NO: 98 and the        second nucleic acid comprising SEQ ID NO: 99;    -   5) the first nucleic acid comprising SEQ ID NO: 98 and the        second nucleic acid comprising SEQ ID NO: 100;    -   6) the first nucleic acid comprising SEQ ID NO: 98 and the        second nucleic acid comprising SEQ ID NO: 101;    -   7) the first nucleic acid comprising SEQ ID NO: 107 and the        second nucleic acid comprising SEQ ID NO: 110;    -   8) the first nucleic acid comprising SEQ ID NO: 107 and the        second nucleic acid comprising SEQ ID NO: 111;    -   9) the first nucleic acid comprising SEQ ID NO: 108 and the        second nucleic acid comprising SEQ ID NO: 110;    -   10) the first nucleic acid comprising SEQ ID NO: 108 and the        second nucleic acid comprising SEQ ID NO: 111;    -   11)the first nucleic acid comprising SEQ ID NO: 109 and the        second nucleic acid comprising SEQ ID NO: 110;    -   12) the first nucleic acid comprising SEQ ID NO: 109 and the        second nucleic acid comprising SEQ ID NO: 111;    -   13) the first nucleic acid comprising SEQ ID NO: 116 and the        second nucleic acid comprising SEQ ID NO: 110;    -   14) the first nucleic acid comprising SEQ ID NO: 116 and the        second nucleic acid comprising SEQ ID NO: 111;    -   15) the first nucleic acid comprising SEQ ID NO: 117 and the        second nucleic acid comprising SEQ ID NO: 110;    -   16) the first nucleic acid comprising SEQ ID NO: 117 and the        second nucleic acid comprising SEQ ID NO: 111;    -   17) the first nucleic acid comprising SEQ ID NO: 112 and the        second nucleic acid comprising SEQ ID NO: 114;    -   18) the first nucleic acid comprising SEQ ID NO: 112 and the        second nucleic acid comprising SEQ ID NO: 115;    -   19) the first nucleic acid comprising SEQ ID NO: 113 and the        second nucleic acid comprising SEQ ID NO: 114; or    -   20) the first nucleic acid comprising SEQ ID NO: 113 and the        second nucleic acid comprising SEQ ID NO: 115.

In one respect, the invention provides an expression vector composition,which comprises:

-   -   (I) a first expression vector comprising a nucleotide sequence        selected from the group consisting of SEQ ID NOs: 97, 98, 107,        108, 109, 112, 113, 116 and 117; and    -   (II) a second expression vector comprising a nucleotide        sequences selected from the group consisting of SEQ ID NOs: 99,        100, 101, 110, 111, 114 and 115.

In some embodiments, the invention provides the expression vectorcomposition, which comprises:

-   -   1) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 97 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 99;    -   2) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 97 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 100;    -   3) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 97 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 101;    -   4) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 98 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 99;    -   5) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 98 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 100;    -   6) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 98 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 101;    -   7) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 107 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 110;    -   8) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 107 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 111;    -   9) a first expression vector comprising a nucleotide sequence of        SEQ ID NO: 108 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 110;    -   10) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 108 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 111;    -   11) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 109 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 110;    -   12) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 109 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 111;    -   13) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 116 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 110;    -   14) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 116 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 111;    -   15) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 117 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 110;    -   16) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 117 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 111;    -   17) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 112 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 114;    -   18) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 112 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 115;    -   19) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 113 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 114; or    -   20) a first expression vector comprising a nucleotide sequence        of SEQ ID NO: 113 and a second expression vector comprising a        nucleotide sequence of SEQ ID NO: 115.

In another respect, the invention provides an expression vector, whichcomprises:

-   -   (I) a first nucleic acid sequence comprising a nucleotide        sequence selected from the group consisting of SEQ ID NOs: 97,        98, 107, 108, 109, 112, 113, 116 and 117; and    -   (II) a second nucleic acid sequence comprising a nucleotide        sequence selected from the group consisting of SEQ ID NOs: 99,        100, 101, 110, 111, 114 and 115.

In some embodiments, the expression vector, comprises:

-   -   1) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 97 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 99;    -   2) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 97 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 100;    -   3) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 97 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 101;    -   4) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 98 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 99;    -   5) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 98 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 100;    -   6) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 98 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 101;    -   7) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 107 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 110;    -   8) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 107 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 111;    -   9) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 108 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 110;    -   10) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 108 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 111;    -   11) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 109 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 110;    -   12) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 109 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 111;    -   13) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 116 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 110;    -   14) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 116 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 111;    -   15) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 117 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 110;    -   16) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 117 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 111;    -   17) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 112 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 114;    -   18) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 112 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 115;    -   19) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 113 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 114; or    -   20) a first nucleic acid sequence comprising a nucleotide        sequence of SEQ ID NO: 113 and a second nucleic acid sequence        comprising a nucleotide sequence of SEQ ID NO: 115.

In yet another respect, the invention provides a cell comprising any oneof the expression vector composition.

In one respect, the invention provides a method of preparing theisolated antibody or antigen-binding fragment thereof, comprisingexpressing the isolated antibody or antigen-bind thereof in the cell,and isolating the isolated antibody or antigen-binding fragment from thecell.

In another respect, the invention provides a pharmaceutical compositioncomprising the isolated antibody or antigen-binding fragment thereof,and a pharmaceutically acceptable excipient.

In yet another respect, the invention provides a kit comprising theisolated antibody or antigen-binding fragment thereof.

In one respect, the invention provides a method of treating tumorcomprising administering a subject in need a therapeutically effectiveamount of the isolated antibody or antigen-binding fragment thereof, orthe pharmaceutical composition. In some embodiments, the tumor isselected from solid tumor or hematological tumor. In some embodiments,the tumor is selected from bladder cancer, breast cancer, cervicalcancer, ovarian cancer, prostate cancer, testicular cancer, esophagealcancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer,colon cancer, renal cancer, head and neck cancer, lung cancer (smallcell lung cancer or non-small cell lung cancer), stomach cancer, bonecancer, liver cancer, thyroid cancer, skin cancer, central nervoussystem tumor, lymphoma, leukemia, myeloma, sarcoma, and virus-associatedcancer.

In another respect, the invention provides use of the isolated antibodyor antigen-binding fragment thereof, or the pharmaceutical compositionfor the manufacture of a medicament for a treatment of tumor. In someembodiments, the tumor is selected from solid tumor or hematologicaltumor. In some embodiments, the tumor is selected from bladder cancer,breast cancer, cervical cancer, ovarian cancer, prostate cancer,testicular cancer, esophageal cancer, gastrointestinal cancer,pancreatic cancer, colorectal cancer, colon cancer, renal cancer, headand neck cancer, lung cancer (small cell lung cancer or non-small celllung cancer), stomach cancer, bone cancer, liver cancer, thyroid cancer,skin cancer, central nervous system tumor, lymphoma, leukemia, myeloma,sarcoma, and virus-associated cancer.

In yet another respect, the invention provides the isolated antibody orantigen-binding fragment thereof, or the pharmaceutical composition foruse in a treatment of tumor. In some embodiments, the tumor is selectedfrom solid tumor or hematological tumor. In some embodiments, the canceris selected from bladder cancer, breast cancer, cervical cancer, ovariancancer, prostate cancer, testicular cancer, esophageal cancer,gastrointestinal cancer, pancreatic cancer, colorectal cancer, coloncancer, renal cancer, head and neck cancer, lung cancer (small cell lungcancer or non-small cell lung cancer), stomach cancer, bone cancer,liver cancer, thyroid cancer, skin cancer, central nervous system tumor,lymphoma, leukemia, myeloma, sarcoma, and virus-associated cancer.

TABLE I Description of the antibody sequence listing of the inventionSEQ ID Sequence NO: Description Sequence 1 huCD73WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGG VARLFTKVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEGLI EPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDE ITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYP FIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLD NYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDE RNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDR VVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMK VIYPAVEGRIKAHHHHHHHHHH 2 cynoCD73WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGG VARLFTKVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEGLI EPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDE ITALQPEVDKLKTLNVNKIIALGHSGFETDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYP FIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLD NYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHADEMEWNHVSMCILNGGGIRSPIDE RNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDR VVKLDVLCTKCRVPSYDPLKMDEIYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMK VIYPAVEGRIKAHHHHHHHHHH 3 S1B5 VH aaEVQLKESGAELVKPGASVKISCKATGYTFTGYWI EWVKQRPGRGLEWIGEILPGSDITNYNEKFKGKATITADTSSNTAYMQLSSLTTEDSAIYYCARRGYD ETGYAMDYWGQGTSVTVSS 4 S1B5 VH ntGAGGTGCAGCTGAAGGAGTCTGGGGCTGAGCTGG TGAAGCCTGGGGCCTCAGTGAAGATTTCCTGCAAAGCTACTGGCTATACATTCACTGGCTACTGGATA GAGTGGGTAAAGCAGAGGCCTGGACGTGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGATAT TACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACAATCACTGCAGATACATCCTCCAACACAGCCT ACATGCAACTCAGCAGCCTGACAACTGAGGACTCTGCCATCTATTACTGTGCAAGAAGGGGTTACGAC GAGACGGGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACAGTCTCCTCA 5 S1B5 VH GYWIE CDR1 6 S1B5 VH EILPGSDITNYNEKFKGCDR2 7 S1B5 VH RGYDETGYAMDY CDR3 8 S1B5 VL aaDIVMTQSPASLAVSLGQRATISCKASQSVDYDGD SYMNWYQQKPGQPPKLLIHAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAAVYFCQQSKEVPWTF GEGTKLEIK 9 S1B5 VL ntGACATTGTGATGACCCAATCTCCAGCTTCTTTGG CTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGAT AGCTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCCATGCTGCATCCAA TCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCC ATCCTGTGGAGGAAGAGGATGCTGCAGTGTATTTCTGTCAGCAAAGTAAGGAGGTTCCGTGGACGTTC GTGGAAGGGACCAAGCTGGAAATCAAA 10S1B5 VL KASQSVDYDGDSYMN CDR1 11 S1B5 VL AASNLES CDR2 12 S1B5 VLQQSKEVPWT CDR3 13 S1B5 hIgG1 EVQLKESGAELVKPGASVKISCKATGYTFTGYWIfull heavy EWVKQRPGRGLEWIGEILPGSDITNYNEKFKGKA chainTITADTSSNTAYMQLSSLTTEDSAIYYCARRGYD ETGYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 14 S1B5DIVMTQSPASLAVSLGQRATISCKASQSVDYDGD hkappa fullSYMNWYQQKPGQPPKLLIHAASNLESGIPARFSG light chainSGSGTDFTLNIHPVEEEDAAVYFCQQSKEVPWTF GEGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 15 S1B5 hIgG2 EVQLKESGAELVKPGASVKISCKATGYTFTGYWIfull heavy EWVKQRPGRGLEWIGEILPGSDITNYNEKFKGKA chainTITADTSSNTAYMQLSSLTTEDSAIYYCARRGYD ETGYAMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVL TVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 16 JB24ChiQVQLQQSGLELVKPGASVKMSCKASGYTFTDYNM VH aaHWVKQSHGKSLEWIGYIKPHNGGTTYNPKFEGKA TLTVNKSSSTAYMELRSLTSEDSAVYYCVRCDFLYWYFDVWGTGTTVTVSS 17 JB24Chi CAGGTCCAACTGCAGCAGTCTGGACTTGAGCTGG VH ntTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAA GGCTTCTGGATACACATTCACTGACTACAACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTG AGTGGATTGGATATATTAAGCCTCACAATGGTGGTACTACCTACAACCCGAAGTTCGAGGGCAAGGCC ACATTGACTGTAAACAAGTCTTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCGGAGGATTC TGCAGTCTATTACTGTGTAAGATGCGATTTTCTCTACTGGTATTTCGATGTCTGGGGCACAGGGACCA CGGTCACCGTCTCCTCA 18 JB24Chi DYNMHVH CDR1 19 JB24Chi YIKPHNGGTTYNPKFEG VH CDR2 20 JB24Chi CDFLYWYFDVVH CDR3 21 JB24Chi DIVMTQSPAIMSASLGERVTMTCTASSSVSSSYL VL aaHWYQQKPGSSPKLWIYSTSNLASGVPGRFSGSGS GTSYSLTISSMEAEDAATYYCHQYHRSPLTFGAGTKLEMK 22 JB24Chi GACATTGTGATGACCCAGTCTCCAGCAATCATGT VL ntCTGCATCTCTAGGGGAACGGGTCACCATGACCTG CACTGCCAGCTCAAGTGTAAGTTCCAGTTACTTGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCA AACTCTGGATTTATAGCACATCCAACCTGGCTTCTGGAGTCCCAGGTCGCTTCAGTGGCAGTGGGTCT GGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCACCA GTATCATCGTTCCCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAAATGAAA 23 JB24Chi TASSSVSSSYLH VL CDR1 24 JB24Chi STSNLASVL CDR2 25 JB24Chi HQYHRSPLT VL CDR3 26 JB24ChiQVQLQQSGLELVKPGASVKMSCKASGYTFTDYNM hIgG1 fullHWVKQSHGKSLEWIGYIKPHNGGTTYNPKFEGKA  heavy chainTLTVNKSSSTAYMELRSLTSEDSAVYYCVRCDFL  YWYFDVWGTGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 27 JB24ChiQVQLQQSGLELVKPGASVKMSCKASGYTFTDYNM  hIgG2 fullHWVKQSHGKSLEWIGYIKPHNGGTTYNPKFEGKA heavy chainTLTVNKSSSTAYMELRSLTSEDSAVYYCVRCDFL YWYFDVWGTGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 28 JB24ChiDIVMTQSPAIMSASLGERVTMTCTASSSVSSSYL hkappa fullHWYQQKPGSSPKLWIYSTSNLASGVPGRFSGSGS  light chainGTSYSLTISSMEAEDAATYYCHQYHRSPLTFGAG TKLEMKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 29 66F2A8D6 QVHLQQSGAELAKPGASVNLSCKASGYAFTSYWM VH aaHWVKQRPGQGLEWIGYINPSSGLAKYNQKFKDKA TLTTDKSSNTAYMQLSSLTYDDSAVYYCGRWLLSAWFAYWGQGTLVTVSA 30 66F2A8D6 CAGGTCCACCTGCAGCAGTCTGGGGCTGAACTGG VH ntCAAAACCTGGGGCCTCAGTGAACCTGTCCTGCAA GGCTTCTGGCTACGCCTTTACTAGTTACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGG AATGGATTGGATACATTAATCCTAGCAGTGGTCTTGCTAAGTATAATCAGAAGTTCAAAGACAAGGCC ACATTGACTACAGACAAATCTTCCAACACAGCCTACATGCAACTGAGCAGCCTGACATATGACGACTC TGCAGTCTATTACTGTGGAAGATGGTTACTTTCGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGG TCACTGTCTCTGCA 31 66F2A8D6 SYWMHVH CDR1 32 66F2A8D6 YINPSSGLAKYNQKFKD VH CDR2 33 66F2A8D6 WLLSAWFAYVH CDR3 34 66F2A8D6 DIKMTQSPSSIYASLGERVTITCKASQGINTYLS VL aaWFQQKPGKSPKTLIYRANILVDGVPSRFSGSGSG QDYSLTINSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIK 35 66F2A8D6 GACATCAAGATGACCCAGTCTCCATCTTCCATAT VL ntATGCATCTCTAGGAGAGAGAGTCACTATCACTTG CAAGGCGAGTCAGGGCATTAATACCTATTTAAGCTGGTTCCAGCAGAAACCAGGAAAATCTCCTAAGA CCCTGATCTATCGTGCAAACATCTTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGG CAAGATTATTCTCTCACCATCAACAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACAGTA TGATGAGTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA 36 66F2A8D6 KASQGINTYLS VL CDR1 37 66F2A8D6 RANILVDVL CDR2 38 66F2A8D6 LQYDEFPYT VL CDR3 39 66-hIgG2QVHLQQSGAELAKPGASVNLSCKASGYAFTSYWM full heavyHWVKQRPGQGLEWIGYINPSSGLAKYNQKFKDKA chainTLTTDKSSNTAYMQLSSLTYDDSAVYYCGRWLLS AWFAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVV HQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 40 66-hkappaDIKMTQSPSSIYASLGERVTITCKASQGINTYLS full lightWFQQKPGKSPKTLIYRANILVDGVPSRFSGSGSG chainQDYSLTINSLEYEDMGIYYCLQYDEFPYTFGGGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 41 94A12G11F2 QVQLQQSGAELAKPGASVKLSCKASGYTFTSYWM VH aaHWVKQRPGQGLEWIGYINPSSGYTKSNQKFKDKA TLTADKSSSTAYMQLSSLTYEDSAVYYCGRWLLSAWFAYWGQGTLVTVSA 42 94A12G11F2 CAGGTCCAGCTGCAGCAGTCTGGGGCTGAACTGG VH ntCAAAACCTGGGGCCTCAGTGAAGCTGTCCTGCAA GGCTTCTGGCTACACCTTTACTAGTTACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGG AATGGATTGGATACATTAATCCTAGCAGTGGTTATACTAAGTCCAATCAGAAGTTCAAGGACAAGGCC ACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTGAGCAGCCTGACATATGAGGACTC TGCAGTCTATTACTGTGGAAGATGGTTACTTTCGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGG TCACTGTCTCTGCA 43 94A12G11F2 SYWMHVH CDR1  44 94A12G11F2 YINPSSGYTKSNQKFKD VH CDR2  45 94A12G11F2WLLSAWFAY VH CDR3 46 94A12G11F2 DIRMTQSPSSMYASLGERVTITCKASQDINTYLS VL aaWFQQKPGKSPKSLIYRSNILVDGVPSRFSGSGSG QDYSLTISSLEYEDMGIYYCLQYDDFPYTFGGGTKLEIK 47 94A12G11F2 GACATCAGGATGACCCAGTCTCCATCTTCCATGT VL ntATGCATCTCTAGGAGAGAGAGTCACTATCACTTG CAAGGCGAGTCAGGACATTAATACCTATTTAAGCTGGTTCCAGCAGAAACCAGGAAAATCTCCTAAGT CCCTGATCTATCGCTCAAACATCTTGGTAGATGGGGTCCCATCAAGATTCAGTGGCAGTGGATCTGGT CAAGATTATTCTCTCACCATCAGCAGCCTGGAGTATGAGGATATGGGAATTTATTATTGTCTACAGTA TGATGACTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA 48 94A12G11F2 KASQDINTYLS VL CDR1 49 94A12G11F2 RSNILVDVL CDR2 50 94A12G11F2 LQYDDFPYT VL CDR3 51 94-hIgG2QVQLQQSGAELAKPGASVKLSCKASGYTFTSYWM full heavyHWVKQRPGQGLEWIGYINPSSGYTKSNQKFKDKA  chainTLTADKSSSTAYMQLSSLTYEDSAVYYCGRWLLS AWFAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVV HQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 52 94-hIgG1QVQLQQSGAELAKPGASVKLSCKASGYTFTSYWM full heavyHWVKQRPGQGLEWIGYINPSSGYTKSNQKFKDKA chainTLTADKSSSTAYMQLSSLTYEDSAVYYCGRWLLS AWFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 53 94-hkappaDIRMTQSPSSMYASLGERVTITCKASQDINTYLS full lightWFQQKPGKSPKSLIYRSNILVDGVPSRFSGSGSG  chainQDYSLTISSLEYEDMGIYYCLQYDDFPYTFGGGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 54 191C3A8B9 QVQLKESGPGLVAPSQSLSITCTVSGFSLTNYGV VH aaHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLS ISKDNSKSQLFLKMNSLQADDTAMYYCARERGSSWGTMDYWGQGTSVTVSS 55 191C3A8B9 CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGG VH ntTGGCGCCCTCACAGAGCCTGTCCATCACTTGCAC TGTCTCTGGGTTTTCATTAACCAACTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGG AGTGGCTGGGAGTAATATGGGCTGGTGGAAGCACAAATTATAATTCGGCTCTCATGTCCAGACTGAGC ATCAGCAAAGACAACTCCAAGAGCCAACTTTTCTTAAAAATGAACAGTCTGCAAGCTGATGACACAGC CATGTACTACTGTGCCAGAGAGAGGGGTAGTAGCTGGGGGACTATGGACTACTGGGGTCAAGGAACCT CAGTCACTGTCTCCTCA 56 191C3A8B9 NYGVHVH CDR1 57 191C3A8B9 VIWAGGSTNYNSALMS VH CDR2  58 191C3A8B9 ERGSSWGTMDYVH CDR3 59 191C3A8B9 QIVLTQSPAIMSASPGEKVTMTCSASSRVSYMHW VL aaYQQKSGTSPKRWIYDTSQLASGVPARFSGSGSGT SYSLTISSMEAEDAATYYCQQWSSNPYTFGGGTKLEMR 60 191C3A8B9 CAAATTGTTCTCACCCAGTCTCCAGCAATCATGT VL ntCTGCATCTCCAGGGGAGAAGGTCACCATGACCTG CAGTGCCAGCTCACGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGAT GGATTTATGACACATCCCAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACC TCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAG TAGTAACCCATACACGTTCGGAGGGGGGACCAAGCTGGAAATGAGA 61 191C3A8B9 SASSRVSYMH VL CDR1 62 191C3A8B9 DTSQLASVL CDR2 63 191C3A8B9 QQWSSNPYT VL CDR3  64 191-hIgG2QVQLKESGPGLVAPSQSLSITCTVSGFSLTNYGV full heavyHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLS  chainISKDNSKSQLFLKMNSLQADDTAMYYCARERGSS WGTMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 65 191-hkappaQIVLTQSPAIMSASPGEKVTMTCSASSRVSYMHW full lightYQQKSGTSPKRWIYDTSQLASGVPARFSGSGSGT chainSYSLTISSMEAEDAATYYCQQWSSNPYTFGGGTK LEMRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 66 JB24H2 VH QVQLVQSGAEVKKPGASVKMSCKASGYTFTDYNMHWVRQSPGKSLEWIGYIKPHNAGTTYNPKFEGRA TLTVDTSASTAYMELSSLRSEDTAVYYCVRSDFLYWYFDVWGQGTTVTVSS 67 JB24H3 VH QVQLVQSGAEVKKPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWIGYIKPHNAGTTYNPKFEGRA TLTVDTSASTAYMELRSLRSEDTAVYYCVRSDFLYWYFDVWGQGTTVTVSS 68 JB24L1 VL DIQMTQSPSSLSASVGDRVTITCTASSSVSSSYLHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCHQYHRSPLTFGAGTKLEIK 69 JB24L2 VL DIQMTQSPSSLSASVGDRVTITCTASSSVSSSYLHWYQQKPGSAPKLWIYSTSNLASGVPSRFSGSGS GTDYTLTISSLQPEDFATYYCHQYHRSPLTFGAGTKLEIK 70 JB24L3 VL DIVMTQSPSSLSASVGDRVTITCTASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPGRFSGSGS GTDYTLTISSLQPEDFATYYCHQYHRSPLTFGAGTKLEIK 71 JB24H2 QVQLVQSGAEVKKPGASVKMSCKASGYTFTDYNM hIgG1 fullHWVRQSPGKSLEWIGYIKPHNAGTTYNPKFEGRA  heavy chainTLTVDTSASTAYMELSSLRSEDTAVYYCVRSDFL  YWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 72 JB24H3QVQLVQSGAEVKKPGASVKMSCKASGYTFTDYNM hIgG1 fullHWVKQSHGKSLEWIGYIKPHNAGTTYNPKFEGRA heavy chainTLTVDTSASTAYMELRSLRSEDTAVYYCVRSDFL  YWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 73 JB24L1DIQMTQSPSSLSASVGDRVTITCTASSSVSSSYL hkappa fullHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGS  light chainGTDFTLTISSLQPEDFATYYCHQYHRSPLTFGAG  TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 74 JB24L2 DIQMTQSPSSLSASVGDRVTITCTASSSVSSSYL  hkappa fullHWYQQKPGSAPKLWIYSTSNLASGVPSRFSGSGS  light chainGTDYTLTISSLQPEDFATYYCHQYHRSPLTFGAG TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 75 JB24L3 DIVMTQSPSSLSASVGDRVTITCTASSSVSSSYL hkappa fullHWYQQKPGSSPKLWIYSTSNLASGVPGRFSGSGS  light chainGTDYTLTISSLQPEDFATYYCHQYHRSPLTFGAG  TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 76 JB94H1 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGYINPSSGYTKSNQKFKDRV TMTADTSTSTAYMELSSLRSEDTAVYYCGRWLLSAWFAYWGQGTLVTVSS 77 JB94H2 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQRPGQGLEWMGYINPSSGYTKSNQKFKDRV TLTADTSTSTAYMELSSLRSEDTAVYYCGRWLLSAWFAYWGQGTLVTVSS 78 JB94H3 VH QVQLQQSGAEVKKPGASVKLSCKASGYTFTSYWMHWVRQRPGQGLEWIGYINPSSGYTKSNQKFKDRA TLTADTSTSTAYMELSSLRSEDTAVYYCGRWLLSAWFAYWGQGTLVTVSS 79 JB94L1 VL DIQMTQSPSSLSASVGDRVTITCKASQDINTYLSWFQQKPGKAPKSLIYRSNILVDGVPSRFSGSGSG QDFTLTISSLQPEDFAIYYCLQYDDFPYTFGQGTKLEIK 80 JB94L3 VL DIQMTQSPSSLSASVGDRVTITCKASQDINTYLSWFQQKPGKSPKSLIYRSNILVDGVPSRFSGSGSG QDYTLTISSLQPEDFAIYYCLQYDDFPYTFGQGTKLEIK 81 JB94H1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWM hIgG1 fullHWVRQAPGQGLEWMGYINPSSGYTKSNQKFKDRV  heavy chainTMTADTSTSTAYMELSSLRSEDTAVYYCGRWLLS  AWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 82 JB94H2QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWM hIgG1 fullHWVRQRPGQGLEWMGYINPSSGYTKSNQKFKDRV heavy chainTLTADTSTSTAYMELSSLRSEDTAVYYCGRWLLS AWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 83 JB94H3QVQLQQSGAEVKKPGASVKLSCKASGYTFTSYWM hIgG1 fullHWVRQRPGQGLEWIGYINPSSGYTKSNQKFKDRA  heavy chainTLTADTSTSTAYMELSSLRSEDTAVYYCGRWLLS AWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGP GSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 84 JB94L1DIQMTQSPSSLSASVGDRVTITCKASQDINTYLS hkappa fullWFQQKPGKAPKSLIYRSNILVDGVPSRFSGSGSG  light chainQDFTLTISSLQPEDFAIYYCLQYDDFPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 85 JB94L3 DIQMTQSPSSLSASVGDRVTITCKASQDINTYLS hkappa fullWFQQKPGKSPKSLIYRSNILVDGVPSRFSGSGSG  light chainQDYTLTISSLQPEDFAIYYCLQYDDFPYTFGQGT  KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 86 JB94H1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWM mIgG1 fullHWVRQAPGQGLEWMGYINPSSGYTKSNQKFKDRV heavy chainTMTADTSTSTAYMELSSLRSEDTAVYYCGRWLLS AWFAYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHT FPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIF PPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQ DWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITV EWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 87 JB94H3QVQLQQSGAEVKKPGASVKLSCKASGYTFTSYWM mIgG1 fullHWVRQRPGQGLEWIGYINPSSGYTKSNQKFKDRA heavy chainTLTADTSTSTAYMELSSLRSEDTAVYYCGRWLLS  AWFAYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHT FPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIF PPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQ DWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITV EWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 88 JB94L1DIQMTQSPSSLSASVGDRVTITCKASQDINTYLS mkappa fullWFQQKPGKAPKSLIYRSNILVDGVPSRFSGSGSG  light chainQDFTLTISSLQPEDFAIYYCLQYDDFPYTFGQGT KLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKD STYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 89 JB94L3 DIQMTQSPSSLSASVGDRVTITCKASQDINTYLS mkappa fullWFQQKPGKSPKSLIYRSNILVDGVPSRFSGSGSG  light chainQDYTLTISSLQPEDFAIYYCLQYDDFPYTFGQGT KLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKD STYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 90 JB94H1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWM hIgG1mtHWVRQAPGQGLEWMGYINPSSGYTKSNQKFKDRV full heavyTMTADTSTSTAYMELSSLRSEDTAVYYCGRWLLS  chainAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 91 JB94H3 QVQLQQSGAEVKKPGASVKLSCKASGYTFTSYWM hIgG1mtHWVRQRPGQGLEWIGYINPSSGYTKSNQKFKDRA full heavyTLTADTSTSTAYMELSSLRSEDTAVYYCGRWLLS  chainAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 92 JB24H2 CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGA VH ntAGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAA GGCCAGCGGCTACACCTTCACCGACTACAACATGCACTGGGTGAGACAGAGCCCCGGCAAGAGCCTGG AGTGGATCGGCTACATCAAGCCCCACAACGCCGGCACCACCTACAACCCCAAGTTCGAGGGCAGAGCC ACCCTGACCGTGGACACCAGCGCCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACAC CGCCGTGTACTACTGCGTAAGAAGCGACTTCCTGTACTGGTACTTCGACGTGTGGGGCCAGGGCACCA CCGTGACCGTGTCCTCA 93 JB24H3CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGA VH ntAGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAA GGCCAGCGGCTACACCTTCACCGACTACAACATGCACTGGGTGAAGCAGAGCCACGGCAAGAGCCTGG AGTGGATCGGCTACATCAAGCCCCACAACGCCGGCACCACCTACAACCCCAAGTTCGAGGGCAGAGCC ACCCTGACCGTGGACACCAGCGCCAGCACCGCCTACATGGAGCTGAGAAGCCTGAGAAGCGAGGACAC CGCCGTGTACTACTGCGTAAGAAGCGATTTTCTCTACTGGTATTTCGATGTCTGGGGCCAGGGCACCA CCGTGACCGTGTCCTCA 94 JB24L1GACATCCAGATGACCCAGAGCCCCAGCAGCCTGA VL ntGCGCCAGCGTGGGCGACAGAGTGACCATCACCTG CACCGCCAGCAGCAGCGTGAGCAGCAGCTACCTGCACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCA AGCTGCTGATCTACAGCACCAGCAACCTGGCCAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGC GGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCACCA GTATCATCGTTCCCCGCTCACGTTCGGCGCCGGCACCAAGCTGGAGATCAAG 95 JB24L2 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGA VL ntGCGCCAGCGTGGGCGACAGAGTGACCATCACCTG CACCGCCAGCAGCAGCGTGAGCAGCAGCTACCTGCACTGGTACCAGCAGAAGCCCGGCAGCGCCCCCA AGCTGTGGATCTACAGCACCAGCAACCTGGCCAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGC GGCACCGACTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCACCA GTATCATCGTTCCCCGCTCACGTTCGGCGCCGGCACCAAGCTGGAGATCAAG 96 JB24L3 GACATCGTGATGACCCAGAGCCCCAGCAGCCTGA VL ntGCGCCAGCGTGGGCGACAGAGTGACCATCACCTG CACCGCCAGCAGCAGCGTGAGCAGCAGCTACCTGCACTGGTACCAGCAGAAGCCCGGCAGCAGCCCCA AGCTGTGGATCTACAGCACCAGCAACCTGGCCAGCGGCGTGCCCGGCAGATTCAGCGGCAGCGGCAGC GGCACCGACTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCACCA GTATCATCGTTCCCCGCTCACGTTCGGCGCCGGCACCAAGCTGGAGATCAAG 97 JB24H2 CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAhIgG1 full AGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAA heavy chainGGCCAGCGGCTACACCTTCACCGACTACAACATG nt CACTGGGTGAGACAGAGCCCCGGCAAGAGCCTGGAGTGGATCGGCTACATCAAGCCCCACAACGCCGG CACCACCTACAACCCCAAGTTCGAGGGCAGAGCCACCCTGACCGTGGACACCAGCGCCAGCACCGCCT ACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCGTAAGAAGCGACTTCCTG TACTGGTACTTCGACGTGTGGGGCCAGGGCACCACCGTGACCGTGTCCTCAGCTAGCACCAAGGGCCC ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGG TCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAG CAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGA AAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACA CCCTGCCCCCATCCCGGGATGAGTTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCC TCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGC AGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCGGGTAAA 98 JB24H3CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGA hIgG1 fullAGAAGCCCGGCGCCAGCGTGAAGATGAGCTGCAA  heavy chainGGCCAGCGGCTACACCTTCACCGACTACAACATG nt CACTGGGTGAAGCAGAGCCACGGCAAGAGCCTGGAGTGGATCGGCTACATCAAGCCCCACAACGCCGG CACCACCTACAACCCCAAGTTCGAGGGCAGAGCCACCCTGACCGTGGACACCAGCGCCAGCACCGCCT ACATGGAGCTGAGAAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCGTAAGAAGCGATTTTCTC TACTGGTATTTCGATGTCTGGGGCCAGGGCACCACCGTGACCGTGTCCTCAGCTAGCACCAAGGGCCC ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGG TCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAG CAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGA AAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACA CCCTGCCCCCATCCCGGGATGAGTTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCC TCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGC AGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCGGGTAAA 99 JB24L1GACATCCAGATGACCCAGAGCCCCAGCAGCCTGA hkappa fullGCGCCAGCGTGGGCGACAGAGTGACCATCACCTG light chainCACCGCCAGCAGCAGCGTGAGCAGCAGCTACCTG  ntCACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCA AGCTGCTGATCTACAGCACCAGCAACCTGGCCAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGC GGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCACCA GTATCATCGTTCCCCGCTCACGTTCGGCGCCGGCACCAAGCTGGAGATCAAGCGTACGGTGGCTGCAC CATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTG CTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAA CTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGC TGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCG CCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 100JB24L2 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGA hkappa fullGCGCCAGCGTGGGCGACAGAGTGACCATCACCTG light chainCACCGCCAGCAGCAGCGTGAGCAGCAGCTACCTG  ntCACTGGTACCAGCAGAAGCCCGGCAGCGCCCCCA AGCTGTGGATCTACAGCACCAGCAACCTGGCCAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGC GGCACCGACTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCACCA GTATCATCGTTCCCCGCTCACGTTCGGCGCCGGCACCAAGCTGGAGATCAAGCGTACGGTGGCTGCAC CATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTG CTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAA CTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGC TGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCG CCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 101JB24L3 GACATCGTGATGACCCAGAGCCCCAGCAGCCTGA hkappa fullGCGCCAGCGTGGGCGACAGAGTGACCATCACCTG light chainCACCGCCAGCAGCAGCGTGAGCAGCAGCTACCTG  ntCACTGGTACCAGCAGAAGCCCGGCAGCAGCCCCA AGCTGTGGATCTACAGCACCAGCAACCTGGCCAGCGGCGTGCCCGGCAGATTCAGCGGCAGCGGCAGC GGCACCGACTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCACCA GTATCATCGTTCCCCGCTCACGTTCGGCGCCGGCACCAAGCTGGAGATCAAGCGTACGGTGGCTGCAC CATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTG CTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAA CTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGC TGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCG CCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 102JB94H1 CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGA VH ntAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAA GGCCAGCGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAGACAGGCCCCCGGCCAGGGCCTGG AGTGGATGGGCTACATCAACCCCAGCAGCGGCTACACCAAGAGCAACCAGAAGTTCAAGGACAGAGTG ACCATGACCGCCGACACCAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACAC CGCCGTGTACTACTGCGGAAGATGGTTACTTTCGGCCTGGTTTGCTTACTGGGGCCAGGGCACCCTGG TGACCGTGAGCAGC 103 JB94H2CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGA VH ntAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAA GGCCAGCGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAGACAGAGACCCGGCCAGGGCCTGG AGTGGATGGGCTACATCAACCCCAGCAGCGGCTACACCAAGAGCAACCAGAAGTTCAAGGACAGAGTG ACCCTGACCGCCGACACCAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACAC CGCCGTGTACTACTGCGGCAGATGGCTGCTGAGCGCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGG TGACCGTGAGCAGC 104 JB94H3CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGGTGA VH ntAGAAGCCCGGCGCCAGCGTGAAGCTGAGCTGCAA GGCCAGCGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAGACAGAGACCCGGCCAGGGCCTGG AGTGGATCGGCTACATCAACCCCAGCAGCGGCTACACCAAGAGCAACCAGAAGTTCAAGGACAGAGCC ACCCTGACCGCCGACACCAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACAC CGCCGTGTACTACTGCGGCAGATGGCTGCTGAGCGCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGG TGACCGTGAGCAGC 105 JB94L1GACATCCAGATGACCCAGAGCCCCAGCAGCCTGA VL ntGCGCCAGCGTGGGCGACAGAGTGACCATCACCTG CAAGGCCAGCCAGGACATCAACACCTACCTGAGCTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGA GCCTGATCTACAGAAGCAACATCCTGGTGGACGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGCGGC CAGGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCATCTACTACTGCCTACAGTA TGATGACTTTCCGTACACGTTCGGCCAGGGCACCAAGCTGGAGATCAAG 106 JB94L3 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGA VL ntGCGCCAGCGTGGGCGACAGAGTGACCATCACCTG CAAGGCCAGCCAGGACATCAACACCTACCTGAGCTGGTTCCAGCAGAAGCCCGGCAAGAGCCCCAAGA GCCTGATCTACAGAAGCAACATCCTGGTGGACGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGCGGC CAGGACTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCATCTACTACTGCCTACAGTA TGATGACTTTCCGTACACGTTCGGCCAGGGCACCAAGCTGGAGATCAAG 107 JB94H1 CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGA hIgG1 fullAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAA  heavy chainGGCCAGCGGCTACACCTTCACCAGCTACTGGATG nt CACTGGGTGAGACAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTACATCAACCCCAGCAGCGGCTA CACCAAGAGCAACCAGAAGTTCAAGGACAGAGTGACCATGACCGCCGACACCAGCACCAGCACCGCCT ACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCGGAAGATGGTTACTTTCG GCCTGGTTTGCTTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATC GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA AGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACC TTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAG CTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAG TTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCAC ATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGG AGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTC CTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCT CCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCC TGCCCCCATCCCGGGATGAGTTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAT CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAA 108 JB94H2CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGA hIgG1 fullAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAA heavy chainGGCCAGCGGCTACACCTTCACCAGCTACTGGATG nt CACTGGGTGAGACAGAGACCCGGCCAGGGCCTGGAGTGGATGGGCTACATCAACCCCAGCAGCGGCTA CACCAAGAGCAACCAGAAGTTCAAGGACAGAGTGACCCTGACCGCCGACACCAGCACCAGCACCGCCT ACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCGGCAGATGGCTGCTGAGC GCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATC GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA AGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACC TTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAG CTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAG TTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCAC ATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGG AGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTC CTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCT CCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCC TGCCCCCATCCCGGGATGAGTTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAT CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAA 109 JB94H3CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGGTGA hIgG1 fullAGAAGCCCGGCGCCAGCGTGAAGCTGAGCTGCAA heavy chainGGCCAGCGGCTACACCTTCACCAGCTACTGGATG  ntCACTGGGTGAGACAGAGACCCGGCCAGGGCCTGG AGTGGATCGGCTACATCAACCCCAGCAGCGGCTACACCAAGAGCAACCAGAAGTTCAAGGACAGAGCC ACCCTGACCGCCGACACCAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACAC CGCCGTGTACTACTGCGGCAGATGGCTGCTGAGCGCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGG TGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC TCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTG GAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAAT CACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATG CCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGG ACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCA AGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGTTGACCAAGAACCA GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGA GGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 110 JB94L1 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAhkappa full GCGCCAGCGTGGGCGACAGAGTGACCATCACCTG  light chainCAAGGCCAGCCAGGACATCAACACCTACCTGAGC  ntTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGA GCCTGATCTACAGAAGCAACATCCTGGTGGACGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGCGGC CAGGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCATCTACTACTGCCTACAGTA TGATGACTTTCCGTACACGTTCGGCCAGGGCACCAAGCTGGAGATCAAGCGTACGGTGGCTGCACCAT CTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTC CCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGA GCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCC GTCACAAAGAGCTTCAACAGGGGAGAGTGT 111JB94L3 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGA hkappa fullGCGCCAGCGTGGGCGACAGAGTGACCATCACCTG light chainCAAGGCCAGCCAGGACATCAACACCTACCTGAGC  ntTGGTTCCAGCAGAAGCCCGGCAAGAGCCCCAAGA GCCTGATCTACAGAAGCAACATCCTGGTGGACGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGCGGC CAGGACTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCATCTACTACTGCCTACAGTA TGATGACTTTCCGTACACGTTCGGCCAGGGCACCAAGCTGGAGATCAAGCGTACGGTGGCTGCACCAT CTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTC CCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGA GCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCC GTCACAAAGAGCTTCAACAGGGGAGAGTGT 112JB94H1 CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGA mIgG1 fullAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAA heavy chainGGCCAGCGGCTACACCTTCACCAGCTACTGGATG  ntCACTGGGTGAGACAGGCCCCCGGCCAGGGCCTGG AGTGGATGGGCTACATCAACCCCAGCAGCGGCTACACCAAGAGCAACCAGAAGTTCAAGGACAGAGTG ACCATGACCGCCGACACCAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACAC CGCCGTGTACTACTGCGGAAGATGGTTACTTTCGGCCTGGTTTGCTTACTGGGGCCAGGGCACCCTGG TGACCGTGAGCAGCGCCAAGACCACCCCTCCTTCCGTGTATCCTCTGGCTCCAGGATCCGCCGCTCAG ACAAACTCCATGGTGACCCTGGGTTGCCTGGTGAAGGGCTACTTCCCTGAGCCAGTGACCGTGACTTG GAACTCCGGCTCTCTGTCTTCCGGAGTGCACACATTTCCAGCCGTGCTGCAGAGCGACCTGTACACAC TGTCCTCCTCCGTGACCGTGCCTTCTTCCACTTGGCCTTCCGAGACCGTGACTTGCAACGTGGCCCAC CCAGCCTCTTCTACCAAGGTGGACAAGAAGATCGTCCCCCGGGATTGCGGTTGCAAGCCTTGCATTTG CACCGTGCCCGAGGTGTCCTCCGTGTTCATCTTCCCTCCCAAGCCTAAGGACGTGCTGACCATCACCC TGACCCCCAAAGTGACTTGCGTGGTGGTGGACATCTCTAAGGACGACCCCGAGGTGCAGTTCTCTTGG TTCGTGGACGACGTGGAGGTGCACACAGCTCAGACACAGCCCCGGGAGGAGCAGTTCAACTCCACCTT CCGGAGCGTGTCCGAGCTGCCCATCATGCACCAGGATTGGCTGAACGGCAAGGAGTTCAAGTGCCGCG TGAACAGCGCCGCTTTTCCAGCCCCTATCGAGAAGACCATCTCCAAGACCAAGGGCAGGCCCAAGGCT CCTCAGGTGTACACCATCCCTCCCCCTAAGGAGCAGATGGCCAAGGACAAGGTGTCCCTGACTTGCAT GATCACCGACTTCTTCCCCGAGGACATCACAGTCGAGTGGCAGTGGAACGGCCAGCCAGCCGAGAACT ACAAGAACACCCAGCCCATCATGGATACCGACGGCTCTTACTTCGTGTACTCCAAGCTGAACGTGCAG AAGTCCAATTGGGAGGCCGGCAACACCTTCACTTGCTCCGTGCTGCACGAGGGACTGCATAACCACCA CACCGAGAAGTCCCTGTCCCACTCTCCCGGCAAG113 JB94H3 CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGGTGA mIgG1 fullAGAAGCCCGGCGCCAGCGTGAAGCTGAGCTGCAA heavy chainGGCCAGCGGCTACACCTTCACCAGCTACTGGATG nt CACTGGGTGAGACAGAGACCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCAGCGGCTA CACCAAGAGCAACCAGAAGTTCAAGGACAGAGCCACCCTGACCGCCGACACCAGCACCAGCACCGCCT ACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCGGCAGATGGCTGCTGAGC GCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAAGACCACCCCTCCTTC CGTGTATCCTCTGGCTCCAGGATCCGCCGCTCAGACAAACTCCATGGTGACCCTGGGTTGCCTGGTGA AGGGCTACTTCCCTGAGCCAGTGACCGTGACTTGGAACTCCGGCTCTCTGTCTTCCGGAGTGCACACA TTTCCAGCCGTGCTGCAGAGCGACCTGTACACACTGTCCTCCTCCGTGACCGTGCCTTCTTCCACTTG GCCTTCCGAGACCGTGACTTGCAACGTGGCCCACCCAGCCTCTTCTACCAAGGTGGACAAGAAGATCG TCCCCCGGGATTGCGGTTGCAAGCCTTGCATTTGCACCGTGCCCGAGGTGTCCTCCGTGTTCATCTTC CCTCCCAAGCCTAAGGACGTGCTGACCATCACCCTGACCCCCAAAGTGACTTGCGTGGTGGTGGACAT CTCTAAGGACGACCCCGAGGTGCAGTTCTCTTGGTTCGTGGACGACGTGGAGGTGCACACAGCTCAGA CACAGCCCCGGGAGGAGCAGTTCAACTCCACCTTCCGGAGCGTGTCCGAGCTGCCCATCATGCACCAG GATTGGCTGAACGGCAAGGAGTTCAAGTGCCGCGTGAACAGCGCCGCTTTTCCAGCCCCTATCGAGAA GACCATCTCCAAGACCAAGGGCAGGCCCAAGGCTCCTCAGGTGTACACCATCCCTCCCCCTAAGGAGC AGATGGCCAAGGACAAGGTGTCCCTGACTTGCATGATCACCGACTTCTTCCCCGAGGACATCACAGTC GAGTGGCAGTGGAACGGCCAGCCAGCCGAGAACTACAAGAACACCCAGCCCATCATGGATACCGACGG CTCTTACTTCGTGTACTCCAAGCTGAACGTGCAGAAGTCCAATTGGGAGGCCGGCAACACCTTCACTT GCTCCGTGCTGCACGAGGGACTGCATAACCACCACACCGAGAAGTCCCTGTCCCACTCTCCCGGCAAG 114 JB94L1GACATCCAGATGACCCAGAGCCCCAGCAGCCTGA mkappa fullGCGCCAGCGTGGGCGACAGAGTGACCATCACCTG  light chainCAAGGCCAGCCAGGACATCAACACCTACCTGAGC  ntTGGTTCCAGCAGAAGCCCGGCAAGGCCCCCAAGA GCCTGATCTACAGAAGCAACATCCTGGTGGACGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGCGGC CAGGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCATCTACTACTGCCTACAGTA TGATGACTTTCCGTACACGTTCGGCCAGGGCACCAAGCTGGAGATCAAGAGAGCCGACGCCGCTCCTA CAGTGTCTATCTTCCCCCCTTCTTCCGAGCAGCTGACCTCTGGAGGAGCCTCCGTCGTGTGTTTCCTC AACAACTTCTACCCCAAGGACATCAACGTCAAGTGGAAGATCGACGGCTCCGAGAGGCAGAACGGCGT GCTGAACTCTTGGACCGACCAGGACTCCAAGGACTCCACCTACTCCATGTCCTCCACCCTGACCCTGA CCAAGGACGAGTACGAGCGGCACAACTCCTACACTTGCGAGGCTACCCACAAGACCTCTACCTCCCCC ATCGTGAAGAGCTTCAACCGCAACGAGTGT 115JB94L3 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGA mkappa fullGCGCCAGCGTGGGCGACAGAGTGACCATCACCTG  light chainCAAGGCCAGCCAGGACATCAACACCTACCTGAGC  ntTGGTTCCAGCAGAAGCCCGGCAAGAGCCCCAAGA GCCTGATCTACAGAAGCAACATCCTGGTGGACGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGCGGC CAGGACTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCATCTACTACTGCCTACAGTA TGATGACTTTCCGTACACGTTCGGCCAGGGCACCAAGCTGGAGATCAAGAGAGCCGACGCCGCTCCTA CAGTGTCTATCTTCCCCCCTTCTTCCGAGCAGCTGACCTCTGGAGGAGCCTCCGTCGTGTGTTTCCTC AACAACTTCTACCCCAAGGACATCAACGTCAAGTGGAAGATCGACGGCTCCGAGAGGCAGAACGGCGT GCTGAACTCTTGGACCGACCAGGACTCCAAGGACTCCACCTACTCCATGTCCTCCACCCTGACCCTGA CCAAGGACGAGTACGAGCGGCACAACTCCTACACTTGCGAGGCTACCCACAAGACCTCTACCTCCCCC ATCGTGAAGAGCTTCAACCGCAACGAGTGT 116JB94H1 CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGA hIgG1mtAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAA full heavyGGCCAGCGGCTACACCTTCACCAGCTACTGGATG  chain ntCACTGGGTGAGACAGGCCCCCGGCCAGGGCCTGG AGTGGATGGGCTACATCAACCCCAGCAGCGGCTACACCAAGAGCAACCAGAAGTTCAAGGACAGAGTG ACCATGACCGCCGACACCAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACAC CGCCGTGTACTACTGCGGAAGATGGTTACTTTCGGCCTGGTTTGCTTACTGGGGCCAGGGCACCCTGG TGACCGTGAGCAGCGCTAGCACAAAAGGACCTTCCGTGTTTCCTCTGGCTCCTTCTTCTAAGTCTACC AGCGGAGGAACAGCAGCTCTGGGTTGTCTGGTGAAAGATTACTTCCCAGAGCCAGTGACAGTGTCTTG GAATTCAGGAGCTCTGACATCAGGAGTGCATACATTTCCAGCAGTGCTGCAGTCTTCAGGTCTGTATT CTCTGTCCTCAGTGGTGACAGTGCCTTCTTCTTCTCTGGGAACCCAGACCTACATCTGTAACGTGAAC CACAAGCCTTCCAACACCAAGGTGGATAAGAGAGTGGAGCCCAAGTCTTGCGATAAGACCCATACTTG CCCTCCTTGTCCAGCTCCAGAATTTGAAGGAGGACCATCAGTGTTCCTGTTTCCTCCTAAGCCTAAGG ACACCCTGATGATCTCCCGGACCCCAGAAGTGACTTGTGTGGTGGTGGACGTGTCTCACGAAGATCCC GAGGTGAAGTTCAATTGGTACGTGGACGGAGTGGAAGTGCATAACGCTAAGACAAAGCCTAGAGAGGA GCAGTACAACTCCACATACAGAGTGGTGTCAGTGCTGACAGTGCTGCATCAGGATTGGCTGAACGGAA AGGAGTACAAGTGCAAGGTGTCTAACAAGGCTCTGCCAGCTTCTATCGAGAAGACCATCTCCAAGGCT AAGGGACAGCCTAGAGAACCTCAGGTGTACACCCTGCCTCCTTCCCGGGAGGAGATGACAAAGAACCA GGTCTCTCTGACTTGTCTGGTGAAGGGCTTTTACCCTTCCGACATCGCCGTGGAATGGGAATCTAACG GACAGCCAGAGAACAACTACAAGACCACACCTCCAGTGCTGGATTCCGACGGCTCCTTCTTCCTGTAC TCCAAGCTGACCGTGGATAAATCTCGTTGGCAGCAGGGAAACGTGTTCTCTTGTAGCGTGATGCACGA AGCTCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCTCCAGGAAAA 117 JB94H3 CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGGTGA hIgG1mtAGAAGCCCGGCGCCAGCGTGAAGCTGAGCTGCAA full heavyGGCCAGCGGCTACACCTTCACCAGCTACTGGATG  chain ntCACTGGGTGAGACAGAGACCCGGCCAGGGCCTGG AGTGGATCGGCTACATCAACCCCAGCAGCGGCTACACCAAGAGCAACCAGAAGTTCAAGGACAGAGCC ACCCTGACCGCCGACACCAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACAC CGCCGTGTACTACTGCGGCAGATGGCTGCTGAGCGCCTGGTTCGCCTACTGGGGCCAGGGCACCCTGG TGACCGTGAGCAGCGCTAGCACAAAAGGACCTTCCGTGTTTCCTCTGGCTCCTTCTTCTAAGTCTACC AGCGGAGGAACAGCAGCTCTGGGTTGTCTGGTGAAAGATTACTTCCCAGAGCCAGTGACAGTGTCTTG GAATTCAGGAGCTCTGACATCAGGAGTGCATACATTTCCAGCAGTGCTGCAGTCTTCAGGTCTGTATT CTCTGTCCTCAGTGGTGACAGTGCCTTCTTCTTCTCTGGGAACCCAGACCTACATCTGTAACGTGAAC CACAAGCCTTCCAACACCAAGGTGGATAAGAGAGTGGAGCCCAAGTCTTGCGATAAGACCCATACTTG CCCTCCTTGTCCAGCTCCAGAATTTGAAGGAGGACCATCAGTGTTCCTGTTTCCTCCTAAGCCTAAGG ACACCCTGATGATCTCCCGGACCCCAGAAGTGACTTGTGTGGTGGTGGACGTGTCTCACGAAGATCCC GAGGTGAAGTTCAATTGGTACGTGGACGGAGTGGAAGTGCATAACGCTAAGACAAAGCCTAGAGAGGA GCAGTACAACTCCACATACAGAGTGGTGTCAGTGCTGACAGTGCTGCATCAGGATTGGCTGAACGGAA AGGAGTACAAGTGCAAGGTGTCTAACAAGGCTCTGCCAGCTTCTATCGAGAAGACCATCTCCAAGGCT AAGGGACAGCCTAGAGAACCTCAGGTGTACACCCTGCCTCCTTCCCGGGAGGAGATGACAAAGAACCA GGTCTCTCTGACTTGTCTGGTGAAGGGCTTTTACCCTTCCGACATCGCCGTGGAATGGGAATCTAACG GACAGCCAGAGAACAACTACAAGACCACACCTCCAGTGCTGGATTCCGACGGCTCCTTCTTCCTGTAC TCCAAGCTGACCGTGGATAAATCTCGTTGGCAGCAGGGAAACGTGTTCTCTTGTAGCGTGATGCACGA AGCTCTGCACAATCACTACACCCAGAAGTCCCTGTCTCTGTCTCCAGGAAAA 118 66F2A8D6 QVHLQQSGAELAKPGASVNLSCKASGYAFTSYWMmIgG1 full HWVKQRPGQGLEWIGYINPSSGLAKYNQKFKDKA  heavy chainTLTTDKSSNTAYMQLSSLTYDDSAVYYCGRWLLS AWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHT FPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIF PPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQ DWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITV EWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 119 66F2A8D6DIKMTQSPSSIYASLGERVTITCKASQGINTYLS mkappa fullWFQQKPGKSPKTLIYRANILVDGVPSRFSGSGSG  light chainQDYSLTINSLEYEDMGIYYCLQYDEFPYTFGGGT  KLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKD STYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 120 94A12G11F2 QVQLQQSGAELAKPGASVKLSCKASGYTFTSYWM mIgG1HWVKQRPGQGLEWIGYINPSSGYTKSNQKFKDKA full heavyTLTADKSSSTAYMQLSSLTYEDSAVYYCGRWLLS chainAWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQ TNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAH PASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSW FVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKA PQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQ KSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK121 94A12G11F2 DIRMTQSPSSMYASLGERVTITCKASQDINTYLS mkappaWFQQKPGKSPKSLIYRSNILVDGVPSRFSGSGSG full lightQDYSLTISSLEYEDMGIYYCLQYDDFPYTFGGGT chainKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFL NNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSP IVKSFNRNEC 122 191C3A8B9QVQLKESGPGLVAPSQSLSITCTVSGFSLTNYGV mIgG1HWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLS full heavyISKDNSKSQLFLKMNSLQADDTAMYYCARERGSS chainWGTMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAA QTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVA HPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFS WFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPK APQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNV QKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG K123 191C3A8B9 QIVLTQSPAIMSASPGEKVTMTCSASSRVSYMHW mkappaYQQKSGTSPKRWIYDTSQLASGVPARFSGSGSGT full lightSYSLTISSMEAEDAATYYCQQWSSNPYTFGGGTK chainLEMRRADAAPTVSIFPPSSEQLTSGGASVVCFLN NFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPI VKSFNRNEC 124 JB94H1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWM hIgG2 fullHWVRQAPGQGLEWMGYINPSSGYTKSNQKFKDRV heavy chainTMTADTSTSTAYMELSSLRSEDTAVYYCGRWLLS  AWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVV HQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 125 JB94H1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWM hIgG4 fullHWVRQAPGQGLEWMGYINPSSGYTKSNQKFKDRV heavy chainTMTADTSTSTAYMELSSLRSEDTAVYYCGRWLLS  AWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK 126 JB94H3QVQLQQSGAEVKKPGASVKLSCKASGYTFTSYWM hIgG2 fullHWVRQRPGQGLEWIGYINPSSGYTKSNQKFKDRA heavy chainTLTADTSTSTAYMELSSLRSEDTAVYYCGRWLLS  AWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVV HQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 127 JB94H3QVQLQQSGAEVKKPGASVKLSCKASGYTFTSYWM hIgG4 fullHWVRQRPGQGLEWIGYINPSSGYTKSNQKFKDRA heavy chainTLTADTSTSTAYMELSSLRSEDTAVYYCGRWLLS AWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK Notes: Unless specified otherwiseherein, all amino acid numbers are according to the EU index of theKabat system (Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). aa: amino acid, nt:nucleotide.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the binding ability assay of positive clones and solublehuCD73 protein;

FIG. 2 shows the binding ability assay of the positive clones and thenatural CD73 protein;

FIG. 3 showing the enzyme activity blockage of soluble recombinant humanCD73;

FIG. 4 shows the cell-based enzyme activity blocking experiment;

FIG. 5 shows CD4+ T cell proliferation reversed by CD73 antibody;

FIG. 6 shows CD73 enzyme activity of CD4+ T cells blocked by antibody;

FIG. 7 shows IFN-γ releasing from CD4+ T cells reversed by antibody;

FIG. 8 shows CD73 endocytosis mediated by CD73 antibody;

FIG. 9 shows the affinity of humanized antibodies and chimericantibodies detected by ELISA;

FIG. 10 shows the affinity of humanized antibodies and chimericantibodies detected by FACs;

FIG. 11 shows the 5′ exonuclease activity on the cell surface blocked byhumanized antibody;

FIG. 12 shows 5′ ectonucleotidase activity of U87-MG cell blocked byhumanized antibody;

FIG. 13 shows CD4+ T cell proliferation reversed by humanized CD73antibody;

FIG. 14 shows IFN-γ releasing from CD4+ T cells reversed by humanizedCD73 antibody;

FIG. 15A and FIG. 15B show the inhibitory effect of humanized antibodiesagainst the 5′ exonuclease activity of tumors in xenograft animalmodels;

FIG. 16 shows the tumor suppression effect of humanized antibody on A375human melanoma xenograft model.

DETAILED DESCRIPTION

Described herein are isolated antibodies, particularly monoclonalantibodies, e.g., human monoclonal antibodies, which specifically bindto CD73 and thereby reduce CD73 activity (“antagonist anti-CD73antibodies”). In certain embodiments, the antibodies described hereinare derived from specific heavy and light chain germline sequencesand/or comprise specific structural features such as CDR regionscomprising specific amino acid sequences. Provided herein are isolatedantibodies, methods of preparing such antibodies. Also provided hereinare methods of using the antibodies for reducing tumor growth, alone orin combination with other therapeutic agents (e.g., antibodies) and/orcancer therapies. Accordingly, the anti-CD73 antibodies described hereinmay be used in a treatment of a wide variety of therapeuticapplications, including, for example, inhibition of tumor growth,inhibition of metastasis, and enhancement of an immune response againsta tumor.

Definitions

In order that the present description may be more readily understood,certain terms are firstly defined. Additional definitions are set forththroughout the detailed description.

The term “Cluster of Differentiation 73” or “CD73” as used herein refersto an enzyme (nucleotidase) capable of converting extracellularnucleoside 5′ monophosphates to nucleosides, namely converting adenosinemonophosphate (AMP) to adenosine. CD73 is usually found as a dimeranchored to the cell membrane through a glycosylphosphatidylinositol(GPI) bond, has ecto-enzyme activity and plays a role in signaltransduction. The primary function of CD73 is converting extracellularnucleotides (e.g., 5′-AMP) to adenosine, which is a highlyimmunosuppressive molecule. Thus, ecto-5′-nucleotidase catalyzes thedephosphorylation of purine and pyrimidine ribo- and deoxyribonucleosidemonophosphates to the corresponding nucleoside. Although CD73 has broadsubstrate specificity, it prefers purine ribonucleosides.

CD73 is also referred to as ecto-5′nuclease (ecto-5′NT, EC 3.1.3.5). Theterm “CD73” includes any variants or isoforms of CD73 which arenaturally expressed by cells. Accordingly, antibodies described hereinmay cross-react with CD73 from species other than human (e.g.,cynomolgus CD73). Alternatively, the antibodies may be specific forhuman CD73 and may not exhibit any cross-reactivity with other species.CD73 or any variants and isoforms thereof, may either be isolated fromcells or tissues which naturally express them or be recombinantlyproduced using well-known techniques in the art and/or those describedherein.

Two isoforms of human CD73 have been identified, both of which share thesame N-terminal and C-terminal portions. Isoform 1 (Accession No.NP_002517.1; SEQ ID NO: 1) represents the longest protein, consisting of574 amino acids and 9 exons. Isoform 2 (Accession No. NP_001191742.1)encodes a shorter protein, consisting of 524 amino acids, lacking aminoacids 404-453. Isoform 2 lacks an alternate in-frame exon, resulting ina transcript with only 8 exons, but with the same N- and C-terminalsequences.

The cynomolgus (cyno) CD73 protein sequence is provided as SEQ ID NO: 2.The terms cynomolgus and cyno both refer to the Macaca fascicularisspecies and are used interchangeably throughout the description.

The term “antibody” as used herein may include whole antibodies and anyantigen binding fragments (i.e., “antigen-binding portions”) or singlechains thereof. An “antibody” refers, in one embodiment, to aglycoprotein or an antigen binding portion thereof comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as VH) and a heavy chain constant region. Insome certain naturally occurring IgG, IgD and IgA antibodies, the heavychain constant region is comprised of three domains, CH1, CH2 and CH3.In some certain naturally occurring antibodies, each light chain iscomprised of a light chain variable region (abbreviated herein as VL)and a light chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), and regions that are more conserved, termedframework regions (FR), both of which are intermingled arrangement.Herein, the CDRs of the VH region are abbreviated as HCDR, that is, thethree CDRs of the VH region can be abbreviated as HCDR1, HCDR2, andHCDR3; the CDRs of the VL region are abbreviated as LCDR, that is, thethree CDRs of the VL region can be abbreviated as LCDR1, LCDR2. LCDR3.Each VH and VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component of theclassical complement system (C1q).

The heavy chain of an antibody may or may not contain a terminal lysine(K), or a terminal glycine and lysine (GK). Thus, any of the heavy chainsequences and heavy chain constant region sequences provided herein canend in either GK or K, or lack K or GK, regardless of what the lastamino acid of the sequence provides. This is because the terminal lysineand sometimes glycine and lysine are cleaved during expression of theantibody.

Antibodies typically bind specifically to their cognate antigen withhigh affinity, reflected by a dissociation constant (K_(D)) of 10⁻⁷ to10⁻¹¹ M or less. Any K_(D) greater than about 10⁻⁶ M is generallyconsidered to indicate binding nonspecifically. As used herein, anantibody that “binds specifically” to an antigen refers to an antibodythat binds to the antigen and substantially identical antigens with highaffinity, which means having a K_(D) of 10⁻⁷ M or less, preferably 10⁻⁸M or less, even more preferably 5×10⁻⁹ M or less, and most preferablybetween 10⁻⁸ M and 10⁻¹⁰ M or less, but does not bind with high affinityto unrelated antigens. An antigen is “substantially identical” to agiven antigen if it exhibits a high degree of sequence identity to thegiven antigen, for example, if it exhibits at least 80%, at least 90%,at least 95%, at least 97%, or at least 99% or greater sequence identityto the sequence of the given antigen. By way of example, an antibodythat binds specifically to human CD73 may also cross-react with CD73from some non-human primate species (e.g., cynomolgus), but may notcross-react with CD73 from other species, or with an antigen other thanCD73.

An immunoglobulin may be from any of the commonly known isotypes,including but not limited to IgA, secretory IgA, IgG and IgM. The IgGisotype is divided in subclasses in some species: IgG1, IgG2, IgG3 andIgG4 in humans, and IgG1, IgG2a, IgG2b and IgG3 in mice. In certainembodiments, the anti-CD73 antibodies described herein are of the humanIgG1 or IgG2 subtype. Immunoglobulins, e.g., human IgG1, exist inseveral allotypes, which differ from each other in at most a few aminoacids. “Antibody” may include, by way of example, both naturallyoccurring and non-naturally occurring antibodies; monoclonal andpolyclonal antibodies; chimeric and humanized antibodies; human andnonhuman antibodies; wholly synthetic antibodies; and single chainantibodies.

The term “antigen-binding portion” of an antibody, as used herein,refers to one or more fragments of an antibody that retain the abilityto specifically bind to an antigen (e.g., human CD73). It has been shownthat the antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibody,e.g., an anti-CD73 antibody described herein, include (i) a Fabfragment, which is a monovalent fragment consisting of the VL, VH, CLand CH1 domains; (ii) a F(ab′)₂ fragment, which is a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR) or (vii) a combination of two or more isolatedCDRs which may optionally be linked by a synthetic linker. Furthermore,although the two domains of the Fv fragment, VL and VH, are encoded bydifferent genes, they can be linked, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules knownas single chain Fv (scFv); see e.g., Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These and other potential constructs are described at Chan & Carter(2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtainedusing conventional techniques known to those with skill in the art, andthe fragments are screened for utility in the same manner as intactantibodies. Antigen-binding portions can be produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intactimmunoglobulins.

The term “amino acid sequence of conservative modifications form” refersto the amino acid modifications that do not significantly affect oralter the binding characteristics of the antibody containing the aminoacid sequence, and the modifications include amino acid substitutions,additions and deletions. Modifications can be introduced into anantibody of the invention by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis. Conservative amino acidsubstitutions are ones in which the amino acid residue is replaced withan amino acid residue having a similar side chain. Families of aminoacid residues having similar side chains have been defined in the art.These families include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, one or more amino acidresidues within the CDR regions of an antibody of the invention can bereplaced with other amino acid residues from the same side chain familyand the altered antibody can be tested for retained function using thefunctional assays described herein. Preferably, the conservativemodifications are no more than one or two in number.

A “bispecific” or “bifunctional antibody” is an artificial hybridantibody having two different heavy/light chain pairs, giving rise totwo antigen binding sites with specificity for different antigens.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelnyet al., J. Immunol. 148, 1547-1553 (1992).

The term “monoclonal antibody,” as used herein, refers to an antibodythat displays a single binding specificity and affinity for a specificepitope or a composition of antibodies in which all antibodies display asingle binding specificity and affinity for a specific epitope.Typically such monoclonal antibodies will be derived from a singleantibody encoding cell or nucleic acid, and will be propagated withoutintentionally introducing any sequence alterations. Accordingly, theterm “human monoclonal antibody” refers to a monoclonal antibody thathas variable and optional constant regions derived from human germlineimmunoglobulin sequences. In one embodiment, human monoclonal antibodiesare produced by a hybridoma, for example, obtained by fusing a B cellderived from a transgenic or transchromosomal non-human animal (e.g., atransgenic mouse having a genome comprising a human heavy chaintransgene and a light chain transgene), with an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, produced or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialhuman antibody library, and (d) antibodies prepared, expressed, producedor isolated by any other means that involve splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies comprise variable and constant regions that utilizespecific human germline immunoglobulin sequences and are encoded by thegermline genes, but include subsequent rearrangements and mutations thatoccur, for example, during antibody maturation. As known in the art(see, e.g., Lonberg (2005) Nature Biotech. 23(9): 1117-1125), thevariable region contains the antigen binding domain, which is encoded byvarious genes that rearrange to form an antibody specific for aexogenous antigen. In addition to rearrangement, the variable region canbe further modified by multiple single amino acid changes (referred toas somatic mutation or hypermutation) to increase the affinity of theantibody to the exogenous antigen. The constant region will change infurther response to an antigen (i.e., isotype switch). Therefore, therearranged and somatically mutated nucleic acid sequences that encodethe light chain and heavy chain immunoglobulin polypeptides in responseto an antigen may not be identical to the original germline sequences,but instead will be substantially identical or similar (i.e. , have atleast 80% identity).

A “human” antibody (HuMAb) refers to an antibody having variable regionsin which both the framework and CDR regions are derived from humangermline immunoglobulin sequences. Furthermore, if the antibody containsa constant region, the constant region is also derived from humangermline immunoglobulin sequences. The antibodies described herein mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo). However, the term“human antibody”, as used herein, is not intended to include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. The terms “human” antibodies and “fully human” antibodies areused synonymously.

A “humanized” antibody refers to an antibody in which some, most or allof the amino acids outside the CDR domains of a non-human antibody arereplaced with corresponding amino acids derived from humanimmunoglobulins. In one embodiment of an antibody in humanized form,some, most or all of the amino acids outside the CDR domains have beenreplaced with amino acids from human immunoglobulins, whereas some, mostor all amino acids within one or more CDR regions are unchanged. Smalladditions, deletions, insertions, substitutions or modifications ofamino acids are permissible as long as they do not abrogate the abilityof the antibody to bind to a specific antigen. A “humanized” antibodyretains an antigenic specificity similar to that of the originalantibody.

A “chimeric antibody” refers to an antibody in which the variableregions are derived from one species and the constant regions arederived from another species, such as an antibody in which the variableregions are derived from a mouse antibody and the constant regions arederived from a human antibody.

A “modified heavy chain constant region” refers to a heavy chainconstant region comprising the constant domains CH1, hinge, CH2, andCH3, wherein one or more of the constant domains are from a differentisotype (e.g. IgG1, IgG2, IgG3, IgG4). In some embodiments, the modifiedconstant region includes a human IgG2 CH1 domain and a human IgG2 hingefused to a human IgG1 CH2 domain and a human IgG1 CH3 domain. In certainembodiments, such modified constant regions also include amino acidmodifications within one or more of the domains relative to the wildtypeamino acid sequence.

As used herein, “isotype” refers to the antibody class (e.g., IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibody) that isencoded by the heavy chain constant region genes.

“Allotype” refers to naturally occurring variants in a specific isotypegroup, which variants differ in a few amino acids (see, e.g., Jefferiset al. (2009) mAbs 1: 1). Antibodies described herein may be of anyallotype.

Unless specified otherwise herein, all amino acid numbers are accordingto the EU index of the Kabat system (Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).

The terms “an antibody recognizing an antigen” and “an antibody specificfor an antigen” are used interchangeably herein with the term “anantibody which binds specifically to an antigen.”

The term “an isolated antibody,” as used herein, is intended to refer toan antibody that is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds to CD73 is substantially free of antibodies thatspecifically bind antigens other than CD73). An isolated antibody thatspecifically binds to an epitope of CD73 may, however, havecross-reactivity to other CD73 proteins from different species.

As used herein, an antibody that “inhibits CD73” refers to an antibodythat inhibits a biological and/or enzymatic function of CD73. Thesefunctions include, for example, the ability of an antibody to inhibitCD73 enzymatic activity, e.g., CD73-regulated production of adenosine orreduction of cAMP production.

As used herein, an antibody that “internalizes” refers to an antibodythat crosses the cell membrane upon binding to a cell-surface antigen.Internalization includes antibody mediated receptor, e.g., CD73,internalization. In some embodiments, the antibody “internalizes” intocells expressing CD73 at a rate of T_(1/2) equal to about 10 min orless.

An “effector function” refers to the interaction of an antibody Fcregion with an Fc receptor or ligand, or a biochemical event thatresults therefrom. Exemplary “effector functions” include C1q binding,complement dependent cytotoxicity (CDC), Fc receptor binding,FcγR-mediated effector functions such as ADCC and antibody dependentcell-mediated hagocytosis (ADCP), and downregulation of a cell surfacereceptor (e.g., the B cell receptor; BCR). Such effector functionsgenerally require the Fc region to be combined with a binding domain(e.g., an antibody variable domain).

An “Fc receptor” or “FcR” is a receptor that binds to the Fc region ofan immunoglobulin. FcRs that bind to an IgG antibody comprise receptorsof the FcγR family, including allelic variants and alternatively splicedforms of these receptors. The FcγR family consists of three activatingreceptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, andFcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). Variousproperties of human FcγRs are summarized in Table A. The majority ofinnate effector cell types coexpress one or more activating FcγR and theinhibitory FcγRIIB, whereas natural killer (NK) cells selectivelyexpress one activating Fc receptor (FcγRIII in mice and FcγRIIIA inhumans) but does not express the inhibitory FcγRIIB in mice and humans.Human IgG1 binds to most human Fc receptors and is considered that thetypes of activating Fc receptors which it binds to are equivalent tomurine IgG2a.

TABLE A Characteristics of human FcγRs Allelic Affinity for Isotype Fcγvariants human IgG preference Cellular distribution FcγRI None High(K_(D) IgG1 = 3 > 4 >> 2 Monocytes, macrophages, described ~10 nM)activated neutrophils, dentritic cells FcγRIIA H131 Low to IgG1 > 3 >2 > 4 Neutrophils, monocytes, medium macrophages, eosinophils, R131 LowIgG1 > 3 > 4 > 2 dentritic cells, platelets FcγRIIIA V158 Medium IgG1 =3 >> 4 > 2 NK cell, monocytes, F158 Low IgG1 = 3 >> 4 > 2 macrophages,mast cells, eosinophils, dentritic cell FcγRIIB I232 Low IgG1 = 3 = 4 >2 B cells, monocytes, T232 Low IgG1 = 3 = 4 > 2 macrophages, dentriticcells, mast cells

A “hinge”, “hinge domain” or “hinge region” or “antibody hinge region”refers to the domain of a heavy chain constant region that links the CH1domain to the CH2 domain and includes the upper, middle, and lowerportions of the hinge (Roux et al. J. Immunol. 1998 161:4083). The hingeprovides varying levels of flexibility between the binding and effectorregions of an antibody and also provides sites for intermoleculardisulfide bonding between the two heavy chain constant regions. The term“hinge” includes wildtype hinges, as well as variants thereof (e.g.,non-naturally-occurring hinges or modified hinges). For example, theterm “IgG2 hinge” includes wildtype IgG2 hinge, and variants having 1,2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2, or 1 mutations,e.g., substitutions, deletions or additions.

The term “CH1 domain” refers to the heavy chain constant region linkingthe variable domain to the hinge in a heavy chain constant domain. Theterm “CH1 domain” includes wildtype CH1 domains, as well as variantsthereof (e.g., non-naturally-occurring CH1 domains or modified CH1domains). For example, the term “CH1 domain” includes wildtype CH1domains and variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/orat most 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions oradditions.

Exemplary CH1 domains include CH1 domains with mutations that change abiological activity of an antibody, such as ADCC, CDC or half-lifeperiod. Modifications to the CH1 domain that affect a biologicalactivity of an antibody are provided herein.

The term “CH2 domain” refers to the heavy chain constant region linkingthe hinge in a heavy chain constant domain to the CH3 domain. The term“CH2 domain” includes wildtype CH2 domains, as well as variants thereof(e.g., non-naturally-occurring CH2 domains or modified CH2 domains). Forexample, the term “CH2 domain” includes wildtype CH2 domains andvariants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5,4, 3, 2, or 1 mutations, e.g., substitutions, deletions or additions.Exemplary CH2 domains include CH2 domains with mutations that change abiological activity of an antibody, such as ADCC, CDC or half-life.

The term “CH3 domain” refers to the heavy chain constant region that isC-terminal to the CH2 domain in a heavy chain constant domain. The term“CH3 domain” includes wildtype CH3 domains, as well as variants thereof(e.g., non-naturally-occurring CH3 domains or modified CH3 domains). Forexample, the term “CH3 domain” includes wildtype CH3 domains andvariants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5,4, 3, 2, or 1 mutations, e.g., substitutions, deletions or additions.Exemplary CH3 domains include CH3 domains with mutations that change abiological activity of an antibody, such as ADCC, CDC or half-life.Modifications to the CH3 domain that affect a biological activity of anantibody are provided herein.

A “CL domain” refers to the constant domain of a light chain. The term“CL domain” includes wildtype CL domains and variants thereof.

A “native sequence Fc region” or “native sequence Fc” comprises an aminoacid sequence that is identical to the amino acid sequence of an Fcregion found in nature. Native sequence human Fc regions include anative sequence human IgG1 Fc region; native sequence human IgG2 Fcregion; native sequence human IgG3 Fc region; and native sequence humanIgG4 Fc region as well as naturally occurring variants thereof. Nativesequence Fc includes the various allotypes of Fcs (see, e.g., Jefferiset al. (2009) mAbs 1: 1).

The term “epitope” or “antigenic determinant” refers to a site on anantigen (e.g., CD73) to which an immunoglobulin or antibody specificallybinds. Epitopes within protein antigens can be formed both fromcontiguous amino acids (usually a linear epitope) or noncontiguous aminoacids juxtaposed by tertiary folding of the protein (usually aconformational epitope). Epitopes formed from contiguous amino acids aretypically, but not always, retained when exposing to denaturingsolvents, whereas epitopes formed by tertiary folding are typically lostwhen treating with denaturing solvents. An epitope typically includes atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in aunique spatial conformation. Methods for determining what epitopes arebound by a given antibody (i.e., epitope mapping) are well known in theart and include, for example, immunoblotting and immunoprecipitationanalysis, wherein overlapping or contiguous peptides (e.g., from CD73)are tested for reactivity with a given antibody (e.g., anti-CD73antibody). Methods of determining spatial conformation of epitopesinclude techniques in the art and those described herein, for example,x-ray crystallography, 2-dimensional nuclear magnetic resonance andHDX-MS (see, e.g., Epitope Mapping Protocols in Methods in MolecularBiology, Vol. 66, G. E. Morris, Ed. (1996)).

The term “epitope mapping” refers to the process of identification ofthe molecular determinants on the antigen involved in antibody-antigenrecognition.

The term “binds to the same epitope” with reference to two or moreantibodies means that the antibodies bind to the same segment of aminoacid residues, as determined by a given method. Techniques fordetermining whether antibodies bind to the “same epitope on CD73” of theantibodies described herein include, for example, epitope mappingmethods, such as, x-ray analyses of crystals of antigen: antibodycomplexes, which provide atomic resolution of the epitope, andhydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methodsthat monitor the binding of the antibody to antigen fragments (e.g.proteolytic fragments) or to mutated variations of the antigen whereloss of binding due to a modification of an amino acid residue in theantigen sequence is often considered an indication of an epitopecomponent (e.g. alanine scanning mutagenesis—Cunningham & Wells (1985)Science 244: 1081). In addition, computational combinatorial methods forepitope mapping can also be used. These methods rely on the ability ofthe antibody of interest from combinatorial phage display peptidelibraries to affinity isolate specific short peptides.

Antibodies that “compete with another antibody for binding to a target”refer to antibodies that inhibit (partially or completely inhibit) thebinding of another antibody to the target. Whether the two antibodiescompete with each other for binding to a target, i.e., whether and towhat extent one antibody inhibits the binding of another antibody to atarget, may be determined using known competition experiments, such asthose described in the Examples. In certain embodiments, an antibodycompetes with another antibody, and inhibit at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or 100% of the binding. The extent of inhibitionor competition may be different depending on which antibody is the“blocking antibody” (i.e., the cold antibody that is incubated firstwith the target). Competition assays can be conducted as described, forexample, in Ed Harlow and David Lane, Cold Spring Harb Pro toe; 2006;doi: 10.1101/pdb.prot4277 or in Chapter 11 of “Using Antibodies” by EdHarlow and David Lane, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., USA 1999. Competing antibodies bind to the same epitope,the overlapping epitope or the adjacent epitopes (e.g., as evidenced bysteric hindrance).

Other competitive binding assays include: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidinEIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phasedirect labeled assay, solid phase direct labeled sandwich analysis (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress (1988)); solid phase direct label RIA using I-125 label (see Morelet al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidinEIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

As used herein, the terms “specific binding,” “selective binding,”“selectively binds,” and “specifically binds,” refer to antibody bindingto an epitope on a predetermined antigen but not to other antigens.Typically, the antibody (i) binds with an equilibrium dissociationconstant (KD) of approximately less than 10⁻⁷M, such as approximatelyless than 10⁻⁸M, 10⁻⁹M or 10⁻¹⁰M or even lower when determined by, e.g.,surface plasmon resonance (SPR) technology in a BIACORE® 2000 surfaceplasmon resonance instrument using the predetermined antigen, e.g.,recombinant human CD73, as the analyte and the antibody as the ligand,or Scatchard analysis of binding of the antibody to antigen positivecells, and (ii) binds to the predetermined antigen with an affinity thatis at least two-times greater than its affinity for binding to anon-specific antigen (e.g., BSA, casein) other than the predeterminedantigen or a closely-related antigen. Accordingly, unless otherwiseindicated, an antibody that “specifically binds to human CD73” refers toan antibody that binds to soluble or cell bound human CD73 with a KD of10⁻⁷M or less, such as approximately less than 10⁻⁸M, 10⁻⁹M or 10⁻¹⁰M oreven lower. An antibody that “cross-reacts with cynomolgus CD73” refersto an antibody that binds to cynomolgus CD73 with a KD of 10⁻⁷M or less,such as less than 10⁻⁸M, 10⁻⁹M or 10⁻¹⁰M or even lower. In certainembodiments, antibodies that do not cross-react with CD73 from anon-human species exhibit essentially undetectable binding against theseproteins in standard binding assays.

The term “Kassoc” or “Ka”, as used herein, is intended to refer to theassociation rate constant of a specific antibody-antigen interaction,whereas the term “Kdis” or “Kd” as used herein, is intended to refer tothe dissociation rate constant of a specific antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe equilibrium dissociation constant, which is obtained from the ratioof Kd to Ka (i.e,. Kd/Ka) and is expressed as a molar concentration (M).K_(D) values of antibodies can be determined using methods wellestablished in the art. A preferred method for determining the K_(D) ofan antibody is to analyze by using surface plasmon resonance, preferablyusing a biosensor system such as a Biacore® surface plasmon resonancesystem or flow cytometry and Scatchard.

The term “EC50” in the context of an in vitro or in vivo assay using anantibody or antigen binding fragment thereof refers to the concentrationof an antibody or an antigen-binding portion thereof that induces aresponse that is 50% of the maximal response, i.e., halfway between themaximal response and the baseline.

A “rate of internalization” of an antibody or of a receptor, e.g., CD73,as mediated by the antibody, e.g., an anti-CD73 antibody, may berepresented, e.g., by T_(1/2) of internalization, e.g., as shown in theExamples. A rate of internalization of an anti-CD73 antibody may beenhanced or increased by at least 10%, 30%, 50%, 75%, 2 times, 3 times,5 times or more, resulting in a reduction of the T_(1/2) by at least10%, 30%, 50%, 75%, 2 times, 3 times, 5 times or more by changing theheavy chain constant region of the antibody to a modified heavy chainconstant region, e.g., one that contains an IgG2 hinge and IgG2 CH1domain. For example, instead of having a T_(1/2) of 10 minutes, amodified heavy chain constant region may increase the rate ofinternalization and thereby reduce the T_(1/2) to 5 minutes (i.e., a twotimes increase in rate of internalization or a two times decrease inT_(1/2)). “T_(1/2)” is defined as the time at which half of the maximalinternalization is achieved, as measured from the time that the antibodyis added to the cells. The maximal level of internalization can be thelevel of internalization at the plateau of a graph representing theinternalization plotted against antibody concentrations. A modifiedheavy chain constant region may increase the maximal level ofinternalization of an antibody by at least 10%, 30%, 50%, 75%, 2 times,3 times, 5 times or more. Another way of comparing internalizationefficacies of different antibodies, such as an antibody with, and thesame antibody without, a modified heavy chain constant region, is bycomparing their level of internalization at a given antibodyconcentration (e.g., 100 nM) or at a given time (e.g., 2 minutes, 5minutes, 10 minutes or 30 minutes). Comparing levels of internalizationcan also be done by comparing the EC50 levels of internalization.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

A “polypeptide” refers to a chain comprising at least two consecutivelylinked amino acid residues, with no upper limit on the length of thechain. One or more amino acid residues in the protein may contain amodification such as, but not limited to, glycosylation, phosphorylationor a disulfide bond. A “protein” may comprise one or more polypeptides.

The term “nucleic acid molecule,” as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may be a singlechain or a double chain, and may be cDNA. Also provided are“conservative sequence modifications” of the sequences set forth in SEQID NOs described herein, i.e., nucleotide and amino acid sequencemodifications which do not abrogate the binding of the antibody encodedby the nucleotide sequence or containing the amino acid sequence to theantigen. Such conservative sequence modifications include conservativenucleotide and amino acid substitutions, as well as, nucleotide andamino acid additions and deletions. For example, modifications can beintroduced into SEQ ID NOs described herein by standard techniques knownin the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative sequence modifications include conservativeamino acid substitutions, in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in an anti-CD73 antibody is preferably replaced withanother amino acid residue from the same side chain family. Methods ofidentifying nucleotide and amino acid conservative substitutions that donot eliminate antigen binding are well-known in the art (see, e.g.,Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. ProteinEng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA94:412-417 (1997)). Alternatively, in another embodiment, mutations canbe introduced randomly along all or part of an anti-CD73 antibodyencoding sequence, such as by saturation mutagenesis, and the resultingmodified anti-CD73 antibodies can be screened through improved bindingactivity.

For nucleic acids, the term “substantial identity ” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial identity exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the chain.

For polypeptides, the term “substantial identity” indicates that twopolypeptides, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate amino acid insertions ordeletions, in at least about 80% of the amino acids, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of theamino acids.

The identity % between two sequences is a function of the number ofidentical positions shared by the sequences when the sequences areoptimally aligned (i.e., identity %=number of identical positions/totalnumber of positions×100), with optimal alignment determined taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences. The comparison ofsequences and determination of percent identity between two sequencescan be accomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the algorithm ofNeedleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences described herein can further beused as a “query sequence” to perform searches against public databasesto, for example, identify related sequences. Such searches can beperformed with the NBLAST and XBLAST programs (version 2.0) of Altschul,et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences identical to the nucleic acid molecules describedherein. BLAST protein searches can be performed with the XBLAST program,score=50, wordlength=3 to obtain amino acid sequences identical to theprotein molecules described herein. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be used as described in Altschulet al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

These nucleic acids may be present in whole cells, in a cell lysate, orin a partially purified or substantially pure form. The nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids (e.g., the other parts of the chromosome) or proteins, bystandard techniques including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. Current Protocols in MolecularBiology, Greene Publishing and Wiley Interscience, New York (1987).

Nucleic acids, e.g., cDNA, may be mutated in accordance with standardtechniques to provide gene sequences. For encoding sequences, thesemutations may affect amino acid sequence as desired. Specifically, DNAsequences substantially identical to or derived from native V, D, J,constant, switches and other such sequences described herein arecontemplated.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is “plasmid,” which refers to acircular double chains DNA loop into which other DNA segments may belinked. Another type of vector is viral vector, wherein other DNAsegments may be linked into the viral genome. Some vectors are capableof autonomous replication in a host cell into which they are introduced(e.g., bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). Other vectors (e.g., non-episomal mammalianvectors) can be integrated into the genome of a host cell whenintroduced into the host cell, and thereby are replicated along with thehost genome. Moreover, some vectors are capable of directing theexpression of genes to which they are operatively linked. Such vectorsare referred to herein as “recombinant expression vectors” (or simply,“expression vectors”). In general, expression vectors used inrecombinant DNA techniques are often in the form of plasmids. In thepresent description, “plasmid” and “vector” may be used interchangeablyas the plasmid is the most commonly used form of vector. However, alsoincluded are other forms of expression vectors, such as viral vectors(e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell that comprises a nucleic acidthat is not naturally present in the cell, and may be a cell into whicha recombinant expression vector has been introduced. It should beunderstood that such terms are intended to refer not only to thespecific subject cell but to the progeny of such a cell. Since certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the term“host cell” as used herein.

As used herein, the term “antigen” refers to any natural or syntheticimmunogenic substance, such as a protein, peptide, or hapten. An antigenmay be CD73 or a fragment thereof.

An “immune response” refers to a biological response in a vertebrate forexogenous agents, such response protects the organism against theseagents and diseases caused by them. An immune response is mediated bythe action of a cell of the immune system (for example, a T lymphocyte,B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mastcell, dendritic cell or neutrophil) and soluble macromolecules producedby any of these cells or the liver (including antibodies, cytokines, andcomplement), the action results in selective targeting, binding to,damage to, destruction of, and/or elimination from the vertebrate's bodyof invading pathogens, cells or tissues infected with pathogens,cancerous or other abnormal cells, or, in cases of autoimmunity orpathological inflammation, normal human cells or tissues. An immuneresponse or reaction includes, e.g., activation or inhibition of a Tcell, e.g., an effector T cell or a Th cell, such as a CD4+ or CD8+ Tcell, or inhibition of a Treg cell.

An “immunomodulator” or “immunoregulator” refers to an agent, e.g., acomponent of a signaling pathway, which may be involved in modulating,regulating, or modifying an immune response. “Modulating,” “regulating,”or “modifying” an immune response refers to any changes in a cell of theimmune system or in the activity of such cell (e.g., an effector Tcell). Such modulation includes stimulation or suppression of the immunesystem which may be manifested by an increase or decrease in the numberof various cell types, an increase or decrease in the activity of thesecells, or any other changes which can occur within the immune system.Both inhibitory and stimulatory immunomodulators have been identified,some of which may have enhanced function in a tumor microenvironment.The immunomodulator may be located on the surface of a T cell. An“immunomodulatory target” or “immunoregulatory target” is animmunomodulator that is targeted for binding by, and whose activity isaltered by the binding of, a substance, agent, moiety, compound ormolecule. Immunomodulatory targets include, for example, receptors onthe surface of a cell (“immunomodulatory receptors”) and receptorligands (“immunomodulatory ligands”).

An increased ability of stimulating an immune response, or the immunesystem can result from an enhanced agonist activity of T cellco-stimulatory receptors and/or an enhanced antagonist activity ofinhibitory receptors. An increased ability of stimulating an immuneresponse or the immune system may be reflected by a time increase of theEC50 or maximal level of activity in an assay that measures an immuneresponse, e.g., an assay that measures changes in cytokine or chemokinerelease, cytolytic activity (determined directly on target cells orindirectly via detecting CD 107a or granzymes) and proliferation. Theability of stimulating an immune response or the immune system activitymay be enhanced by at least 10%, 30%, 50%, 75%, 2 times, 3 times, 5times or more.

“Immunotherapy” refers to the treatment of a subject afflicted with, orat risk of contracting or suffering a recurrence of, a disease by amethod comprising inducing, enhancing, suppressing or otherwisemodifying an immune response.

“Immuno stimulating therapy” or “immuno stimulatory therapy” refers to atherapy that results in increasing (inducing or enhancing) an immuneresponse in a subject for, e.g., treating cancer.

“Potentiating an endogenous immune response” means increasing theeffectiveness or potency of an existing immune response in a subject.This increase in effectiveness and potency may be achieved, for example,by overcoming mechanisms that suppress the endogenous host immuneresponse or by stimulating mechanisms that enhance the endogenous hostimmune response.

“T effector” (“Teff”) cells refers to T cells (e.g., CD4+ and CD8+ Tcells) as well as T helper (Th) cells with cytolytic activities, whichsecrete cytokines and activate and direct other immune cells, but doesnot include regulatory T cells (Treg cells).

As used herein, the term “linkage” refers to the association of two ormore molecules. The linkage can be covalent or non-covalent. The linkagealso can be genetic (i.e., recombinantly fused). Such linkages can beachieved using a wide variety of art recognized techniques, such aschemical coupling and recombinant protein production.

As used herein, “administering” refers to the physical introduction of acomposition comprising a therapeutic agent to a subject, using any ofthe various methods and delivery systems known to those skilled in theart. Preferred routes of administration for antibodies described hereininclude intravenous, intraperitoneal, intramuscular, subcutaneous,spinal or other parenteral routes of administration, for example byinjection or infusion. The phrase “parenteral administration” as usedherein means modes of administration other than enteral and topicaladministration, usually by injection, and includes, but not limited,intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal,intralymphatic, intralesional, intracapsular, intraorbital,intracardiac, intradermal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion, as well as in vivo electroporation.Alternatively, an antibody described herein can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically. Administering can also be performed, forexample, once, a plurality of times, and/or over one or more extendedperiods.

As used herein, the term “T cell-mediated response” refers to a responsemediated by T cells, including effector T cells (e.g., CD8+ cells) andhelper T cells (e.g., CD4+ cells). T cell mediated responses include,for example, T cell cytotoxicity and proliferation.

As used herein, the term “cytotoxic T lymphocyte (CTL) response” refersto an immune response induced by cytotoxic T cells. CTL responses aremediated primarily by CD8+ T cells.

As used herein, the terms “inhibition” or “blocking” (e.g., referring toinhibition/blocking of CD73 binding or activity) are usedinterchangeably and encompass both partial and completeinhibition/blocking.

As used herein, “cancer” refers a broad group of diseases characterizedby the uncontrolled growth of abnormal cells in the body. Sinceunregulated cell division may result in the formation of malignanttumors or cells, they would invade neighboring tissues and maymetastasize to distant parts of the body through the lymphatic system orbloodstream.

The terms “treat,” “treating,” and “treatment,” as used herein, refer toany type of intervention or process performed on, or administering anactive agent to, the subject with the objective of reversing,alleviating, ameliorating, inhibiting, or slowing down or preventing theprogression, development, severity or recurrence of a symptom,complication, condition or biochemical indicia associated with adisease. Prophylaxis refers to administration to a subject who does nothave a disease, to prevent the disease from occurring or minimize itseffects if it does.

A “hematological malignancy” includes lymphoma, leukemia, myeloma orlymphoid malignancy, as well as cancers of the spleen and lymph nodes.Exemplary lymphomas include both B cell lymphomas and T cell lymphomas.B-cell lymphomas include both Hodgkin's lymphomas and most non-Hodgkin'slymphomas. Non-limiting examples of B cell lymphomas include diffuselarge B-cell lymphoma, follicular lymphoma, mucosa-associated lymphatictissue lymphoma, small cell lymphocytic lymphoma (overlaps with chroniclymphocytic leukemia), mantle cell lymphoma (MCL), Burkitt's lymphoma,mediastinal large B cell lymphoma, Waldenstrom macroglobulinemia, nodalmarginal zone B cell lymphoma, splenic marginal zone lymphoma,intravascular large B-cell lymphoma, primary effusion lymphoma,lymphomatoid granulomatosis. Non-limiting examples of T cell lymphomasinclude extranodal T cell lymphoma, cutaneous T cell lymphomas,anaplastic large cell lymphoma, and angioimmunoblastic T cell lymphoma.Hematological malignancies also include leukemia, such as, but notlimited to, secondary leukemia, chronic lymphocytic leukemia, acutemyelogenous leukemia, chronic myelogenous leukemia, and acutelymphoblastic leukemia. Hematological malignancies further includemyelomas, such as, but not limited to, multiple myeloma and smolderingmultiple myeloma. Other hematological and/or B cell- or T-cell-relatedcancers are encompassed by the term hematological malignancy.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve a desired effect. A“therapeutically effective dose” or “therapeutically effective dosage”of a drug or therapeutic agent is any amount of the drug that, when usedalone or in combination with another therapeutic agent, promotes diseaseregression evidenced by a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction. A“prophylactically effective dose” or a “prophylactically effectivedosage” of a drug is an amount of the drug that, when administered aloneor in combination with another therapeutic agent to a subject at risk ofdeveloping a disease or of suffering a recurrence of disease, inhibitsthe development or recurrence of the disease. The ability of atherapeutic or prophylactic agent to promote disease regression orinhibit the development or recurrence of the disease can be evaluatedusing a variety of methods known to those skilled in the art, such as inhuman subjects during clinical trials, in animal model systemspredictive of efficacy in humans, or by assaying the activity of theagent in in-vitro assays.

By way of example, an anti-cancer agent is a drug that slows cancerprogression or promotes cancer regression in a subject. In preferredembodiments, a therapeutically effective amount of the drug promotescancer regression to the point of eliminating the cancer. “Promotingcancer regression” means that administering an effective amount of thedrug, alone or in combination with an anti-neoplastic agent, results ina reduction in tumor growth or size, necrosis of the tumor, a decreasein severity of at least one disease symptom, an increase in frequencyand duration of disease symptom-free periods, a prevention of impairmentor disability due to the disease affliction, or otherwise ameliorationof disease symptoms. Pharmacological effectiveness refers to the abilityof the drug to promote cancer regression in the patient. Physiologicalsafety refers to an acceptably low level of toxicity, or other adversephysiological effects at the cellular, organ and/or organism level(adverse effects) resulting from administration of the drug.

By way of example, for the treatment of tumors, a therapeuticallyeffective dose or dosage of the drug preferably inhibits cell growth ortumor growth by at least about 20%, more preferably by at least about40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects. In themost preferred embodiments, a therapeutically effective dose or dosageof the drug completely inhibits cell growth or tumor growth, i.e.,preferably inhibits cell growth or tumor growth by 100%. The ability ofa compound to inhibit tumor growth can be evaluated using the assaysdescribed infra. Alternatively, this characteristic of a composition canbe evaluated by examining the ability of the compound to inhibit cellgrowth, such inhibition can be measured in vitro by assays known to theskilled practitioner. In other preferred embodiments described herein,tumor regression may be observed and may continue for a period of atleast about 20 days, more preferably at least about 40 days, or evenmore preferably at least about 60 days.

The terms “patient” and “subject” refer to any human or non-human animalthat receives either prophylactic or therapeutic treatment. For example,the methods and compositions described herein can be used to treat asubject having cancer. The term “non-human animal” includes allvertebrates, e.g., mammals and non-mammals, such as non-human primates,sheep, dog, cow, chickens, amphibians, reptiles, etc.

“Pharmaceutically acceptable” is intended to refer to the substance orcomposition that must be chemically and/or toxicologically compatiblewith the other ingredients comprising the formulation and/or the mammalbeing treated with it.

“Pharmaceutically acceptable vectors” encompasses pharmaceuticallyacceptable vectors, excipients, and diluents, and is intended to referto materials, compositions, or vehicles that involve carrying ortransporting pharmaceutical agents from one organ or body part of asubject to another organ or body part of a subject, such as liquid orsolid fillers, diluents, excipients, solvents or encapsulatingmaterials.

EXAMPLES

EXAMPLE 1: Screening and identification of anti-CD73 antibodies

C57/BL6 mice were immunized with human CD73 ecto-domain recombinantprotein (huCD73, SEQ ID NO: 1). Performing the first immunization(intraperitoneal injection) with an emulsion of 50 μg huCD73 proteinplus Freund's complete adjuvant, performing the second immunization(subcutaneous injection)with an emulsion of 25 μg huCD73 protein plusincomplete Freund's adjuvant, performing the third immunization(intraperitoneal injection) with an emulsion of 25 μg huCD73 proteinplus incomplete Freund's adjuvant, performing the fourth immunization(subcutaneous injection) with an emulsion of 25 μg huCD73 protein plusincomplete Freund's adjuvant, and finally, performing the final boostimmunization (intraperitoneal injection) with 50 μg huCD73 protein. Fourdays after the boost, the immune spleen cells were fused with SP2/0cells by electrofusion to prepare hybridoma cells. Mice were immunizedin the same way and phage library antibodies were prepared. Primaryscreening was performed by ELISA and flow cytometry, 32 hybridomaantibodies and 2 phage antibodies were obtained to bind human CD73 andcynomolgus CD73. Then, 293T/17 cells expressing huCD73 (293T/17-huCD73)were used to further screen the blockade enzyme activity, and finally 5clones with function of blockade of CD73 enzyme activity were obtained.

Example 2 Indirect Assay of the Binding of Antibodies to CD73 by ELISA

The indirect ELISA method was applied to evaluate the binding ability ofeach positive clone to soluble huCD73 protein. Soluble huCD73 wascoated, and gradient diluted samples were incubated, then HPR-labeledsecondary antibody was added, finally TMB was added to develop thecolor, and OD450 was read after termination. As shown in FIG. 1, theresult showed that all three cloned antibodies of 66F2A8D6 (SEQ ID NOs:118 and 119), 94A12G11F2 (SEQ ID NOs: 120 and 121), 191C3A8B9 (SEQ IDNOs: 122 and 123) and two phage antibodies of S1B5 (SEQ ID NOs: 13 and14) and JB24Chi (SEQ ID NOs: 26 and 28) bind to soluble recombinant CD73with sub-nanomolar affinity.

TABLE 1 Binding ability of positive clones to soluble huCD73 protein AbBottom Top EC₅₀(nM) 66F2A8D6 0.1646 3.108 0.153 94A12G11F2 0.0566 2.9410.242 191C3A8B9 0.0498 2.748 0.279 S1B5 0.0070 2.926 0.085 JB24Chi0.1360 3.086 0.039

Example 3 Assay of the Binding of Antibodies to Natural CD73 CellSurface by Flow Cytometry

293T/17-huCD73 cells were used to evaluate the binding ability of fiveantibodies to natural CD73 on cell surface. After incubating gradientdiluted antibodies with 293T/17-huCD73 cells, fluorescently labeleddetection antibody was added, the fluorescence intensity value was read,and EC₅₀ was calculated. As shown in FIG. 2, the results showed that allantibodies bind to CD73 on cell surface with nanomolar or sub-nanomolaraffinity.

TABLE 2 Binding ability of positive clones to natural CD73 protein AbBottom Top EC₅₀(nM) 66F2A8D6 0.5465 174.2 1.664 94A12G11F2 1.132 221.72.077 191C3A8B9 2.671 105.4 0.828 S1B5 0.6408 14.66 0.099 JB24Chi −1.46741.98 1.135

Example 4 Evaluation of Blockade of Soluble CD73 Recombinant ProteaseActivity

This assay evaluates the blockade ability of CD73 antibodies to the5′exonuclease of soluble huCD73 recombinant protease. The binding ofantibodies blocks the activity of huCD73 recombinant protein tohydrolysis AMP into adenosine and inorganic phosphate, while thecompetition of AMP with ATP inhibits the ability of luciferase to emitlight. Thus, blocking antibodies attenuate light emission and results inreduced RLU values. As shown in FIG. 3, 66F2A8D6, 94A12G11F2, 191C3A8B9,antibody S1B5 and JB24Chi all have inhibitory effects on the activity ofsoluble CD73 recombinant protein, and the IC₅₀ were at the nanomolar orsubnanomolar level.

TABLE 3 The ability of CD73 antibodies to block soluble CD73 recombinantprotease activity Ab Bottom Top IC₅₀(nM) 66F2A8D6 6.361 86.08 1.95694A12G11F2 6.213 87.77 1.496 191C3A8B9 5.553 93.72 2.407 S1B5 16.18099.47 3.259 JB24Chi 14.710 101.70 0.401

Example 5 Blockade of 5′ Exonuclease on the Cell Surface ActivityAgainst CD73 Antibody

This method was based on 293T/17-huCD73 and 293T/17-cynoCD73 cells toevaluate the blockade ability of CD73 antibodies to 5′exonuclease on thecell surface activity, to further confirm the biochemical activities ofthe five antibodies. As shown in FIG. 4, all antibodies could inhibitthe 5′exonuclease on the cell surface activity. Among them, theinhibitory activity of 191C3A8B9 against huCD73 was weaker than that ofcynomolgus CD73, the others had similar inhibitory activity onhuman/cynomolgus CD73. The IC₅₀ values of all antibodies were10⁻¹¹˜10⁻¹²M (Table 4).

TABLE 4 The ability of CD73 antibodies to block 5 ′exonuclease on thecell surface activity 293T/17-HuCD73 293T/17-cyno CD73 IC₅₀ IC₅₀ AbBottom Top (nM) Bottom Top (nM) 66F2A8D6 3935659 13777015 0.027 25425606682235 0.077 94A12G11F2 4013533 13788420 0.011 2845459 6641976 0.006191C3A8B9 4644137 14192084 0.009 3764123 7369545 0.001 S1B5 853292116191747 0.034 1044320 4968902 0.085 JB24Chi 7418313 16806180 0.0311156967 4317630 0.084

Example 6 Reverse Effect of CD73 Antibody on AMP-Mediated Human CD4+TCell Proliferation Inhibition

Antibodies blocked the enzymatic activity of CD73, and the production ofadenosine was inhibited, thereby releasing the proliferation inhibitionof human CD4+T cells adenosine. This method confirmed the ability ofCD73 antibody to release AMP-mediated CD4+T cell proliferationinhibition in vitro; at the same time, the blockade of CD73 antibodiesto CD4+T cell CD73 was detected by CellTiter-Glo (Promega) reagent; andthe IFN-γ levels in cell culture supernatant were detected by ELISA.

The results showed that 66F2A8D6, 94A12G11F2, S1B5, and JB24Chi canreverse the AMP-mediated CD4+T cell proliferation inhibition (FIG. 5),and the EC₅₀ values were relatively close, all in 10⁻¹¹˜10⁻¹²M (Table 5)

TABLE 5 The ability of CD73 antibodies reversing AMP-mediated humanCD4 + T cell proliferation inhibition Ab Bottom Top EC₅₀(nM) 66F2A8D629.55 69.45 0.010 94A12G11F2 32.63 73.53 0.004 S1B5 27.36 64.14 0.013JB24Chi 26.58 62.33 0.007

Similar to the results of CD4+T cell proliferation experiments,66F2A8D6, 94A12G11F2, 51B5, and JB24Chi all had potent inhibitoryeffects on enzyme activity (FIG. 6), and the inhibitory ability were10⁻¹¹˜10⁻¹²M (Table 6).

TABLE 6 The ability of antibodies blocking CD4 + T cell Ab Bottom TopIC₅₀(nM) 66F2A8D6  8353547 17766093 0.011 94A12G11F2 22404704 469210970.008 S1B5 22907851 25212940 0.048 JB24Chi 45017675 44400168 0.009

ELISA detection kit (Dakewe) was used to detect supernatant IFN-γ, asshown in FIG. 7, 66F2A8D6, 94A12G11F2, S1B5 and JB24Chi can stimulateCD4+T cells to release IFN-γ, and the EC₅₀ of each antibody stimulated Tcells to secrete IFN-γ was in the range of 10⁻¹¹˜10⁻¹²M (Table 7).

TABLE 7 The ablity of antibodies to reverse the release of IFN-γ fromCD4 + T cells Ab Bottom Top EC₅₀(nM) 66F2A8D6  88.7 461.4 0.010194A12G11F2 296.6 794.6 0.0049 S1B5 156.6 576.3 0.0411 JB24Chi 169.1512.1 0.0132

Example 7 Experiment of CD73 Antibody-Mediated CD73 Internalization

66-IgG2 (SEQ ID NOs: 39 and 40), 94-IgG2 (SEQ ID NOs: 51 and 53), S1B5,JB24Chi antibodies mediated internalization effect on MDA-MB-231 cellwas detected using the Fab-ZAP saporin conjugate (Advanced TargetingSystems). As shown in FIG. 8, all four chimeric antibodies mediated theinternalization effect of CD73 in a dose-dependent manner, and the IC₅₀of each antibody was about 10⁻¹¹ M.

TABLE 8 The ability of antibody-mediated internalization of CD73 AbBottom Top IC₅₀(nM) 66-hIgG2 581873 3044537 0.022 94-hIgG2 6496432809795 0.019 S1B5 896346 3084741 0.029 JB24Chi 779820 3197304 0.012

Example 8 Humanization of Antibodies

The CDR and framework regions are numbered, and the amino acids of eachantibody in the CDR regions and framework regions are numbered accordingto the Kabat system (see Table I). Two antibodies 94A12E7D5 and JB24Chiwere humanized by CDR grafting method. The sequence identity andstructural similarity of two murine antibodies and human antibodies wereanalyzed respectively, and the CDRs of the murine antibodies weregrafted to a series of human antibody templates based on the above.After preliminary screening by ELISA, three humanized antibodies ofJB24Chi and three humanized antibodies of 94A12E7D5 were obtained, whichnamed as JB24H2L1 (SEQ ID NOs: 71 and 73), JB24H3L2 (SEQ ID NOs: 72 and74), JB24H3L3 (SEQ ID NOs: 72 and 75), JB94H1L3 (SEQ ID NOs: 81 and 85),JB94H2L1 (SEQ ID NOs: 82 and 84), and JB94H3L3 (SEQ ID NOs: 83 and 85)respectively. Chimeric antibodies consisting of mouse-derivedantibodies' Fv of 94A12G11F and human IgG1 and human kappa constantregions were named JB94Chi (SEQ ID NOS: 52 and 53).

Example 9 The Assay of Affinity of Humanized Antibodies and ChimericAntibodies

The affinity of humanized antibodies was determined by indirect ELISA:96-well ELISA plates were coated with 1 μg/ml huCD73 recombinantprotein, 50 ul/well, washed the plate 3 times with 200 ul PBST per well,blocked with 200 ul/well of 1% BSA for 1 h at room temperature, and theplates were washed 3 times with 200 ul/well of PBST. Incubated withgradient diluted 100 μL/well of antibodies for 1 h at room temperature.After the plates were washed 3 times with 200 ul/well of PBST, 100 ul ofdiluted HRP-labeled secondary antibody at a ratio of 1:5000 was added toeach well, and the plates were incubated for 1 hour at room temperature.The plates were washed 3 times with 200 ul/well of PBST, 100 ul of TMBwas added to each well and reacted for 5 minutes at room temperature inthe dark. Excitation at 530 nm, emission at 590 nm, cut-off at 570 nm,read the OD value. The results were shown in FIG. 9. The affinity ofhumanized antibodies and parent antibodies were similar to the antigen(table 9).

TABLE 9 The affinity of humanized antibodies to soluble huCD73 Ab BottomTop EC₅₀(nM) JB24Chi 128.1 6601 0.0273 JB24H2L1 190.9 6293 0.0248JB24H3L2 89.06 7139 0.0208 JB24H3L3 9.8 7306 0.0228 JB94Chi 152 84740.0357 JB94H1L3 48.7 8288 0.0289 JB94H2L1 −141.9 7111 0.0252 JB94H3L3320 8897 0.0292

Flow cytometry assay: the huCD73 or cynoCD73-expressing 293T/17 cellswere used to evaluate the binding ability of humanized antibodies. 2×10⁶cells resuspended in PBS buffer were distributed into 96-well plates andincubated with gradient diluted humanized antibodies for 1 h atrefrigerator 4° C. or on ice, then the cells was centrifuged for 3 min1500 rpm at 4° C. and washed three times by PBS, and diluted APC-labeledgoat anti-human polyclonal antibody (Biolegend) were were incubated for30 min at refrigerator 4° C. Finally, the cells were washed three timeswith PBS as described above and analyzed on MACSQuant flow cytometer.GraphPad Prism software was used to generate data graphs and statisticalaffinity data. As shown in FIG. 10, humanized antibodies of JB24Chi and94A12G11F had similar EC₅₀ values to their parent antibodies, both inthe 10⁻¹¹ M to 10⁻¹² M (Table 10).

TABLE 10 Affinity of humanized antibodies and chimeric antibodies tohuCD73 and cynoCD73 antigens on cell surface 293T/17-human CD73293T/17-Cyno CD73 Ab Bottom Top EC₅₀ (nM) Bottom Top EC₅₀ (nM) JB24H2L11.639 82.25 2.404 1.401 36.54 1.892 JB24H3L2 1.779 91.74 3.009 0.50948.67 3.593 JB24H3L3 0.131 120.50 7.406 0.917 34.01 3.729 JB24Chi 3.723195.50 5.334 0.709 68.54 6.379 JB94H1L3 3.258 172.50 4.507 1.510 78.705.424 JB94H2L1 0.604 222.80 9.152 0.220 82.03 7.974 JB94H3L3 1.785239.30 15.290 1.395 125.40 9.824 JB94Chi 1.405 246 10.630 1.445 123.908.588

Example 10 Blockade of 5′Exonuclease on the Cell Surface ActivityAgainst Humanized Antibodies

This method uses 293T/17-huCD73 cells to evaluate the AMP hydrolysisinhibited by CD73, to further confirm the biochemical activities ofhumanized antibodies. As shown in FIG. 11, the enzymatic blockingactivities of all humanized antibodies of JB24H3L3, JB94H1L3, 94H1L3hIgG2 (SEQ ID NOs:85 and 124) were in the 10⁻¹¹˜10⁻¹² M levels (Table11).

TABLE 11 Blockade activities of 5 ′exonuclease on the cell surfaceactivity against humanized antibodies 293T/17-human CD73 293T/17-CynoCD73 Cells IC₅₀ IC₅₀ Ab Bottom Top (nM) Bottom Top (nM) JB24H2L1 803954527092351 0.042 6718233 28633058 0.079 JB24H3L2 7836350 27586937 0.0146431345 33705693 0.004 JB24H3L3 7183191 27666447 0.016 7462673 329009760.003 JB24Chi 9147540 26077507 0.006 8819632 26477691 0.005 JB94H1L36727576 21364881 0.042 8000768 30217678 0.017 94H1L3 2016997 109210250.007 1213837 5673059 0.021 hIgG2 JB94H2L1 7233354 22246467 0.0058309853 54243915 0.0001 JB94H3L3 7810738 19948965 0.004 9203356 270049320.002 JB94Chi 7836630 26055691 0.001 9159630 30233789 0.001

In addition, U87-MG cells were used to evaluate hydrolysis of AMPcatalyzed by CD73, and further confirm the biochemical activity ofhumanized antibodies. After U87-MG cells were digested with trypsin, thecell density was adjusted to 4×10⁵ cells/ml with MEM medium, and 50μl/well was added to a 96-well plate. The humanized antibody wasgradient diluted by MEM medium, 50 μl/well was added to the wells, andincubated for 1 h at 37° C. 100 μl of 360 μM AMP was added to each welland incubated for 1 h at 37° C. The plates were then centrifuged for 3min at 1500 rpm, a certain volume of culture supernatants weretransferred to a opaque 96-well flat bottom plate (Costar, 3912), andadded 2× ATP to make the final reaction concentration of 25 μM. Finally,according to Promega instructions, corresponding volume of CellTiter Gloreagent in a ratio of 1:1 was added. After equilibrated for 5 minutes atroom temperature, luminescence value was read on Perkin-Elmer Envisionmicroplate reader and cell CD73 enzyme activity was determined bymeasuring ATP levels. GraphPad Prism software was used to generate datagraphs and statistical enzyme kinetic data. The blocking activities ofhumanized antibodies of JB24Chi and JB94Chi were shown in FIG. 12, bothvalue of IC₅₀ is in the sub-nanomolar class (Table 12).

TABLE 12 Blockade ability of 5′exonuclease activity of U87-MG cellsagainst humanized antibodies Ab Bottom Top IC50(nM) JB24H2L1 579530311954887 0.474 JB24H3L2 4703766 12052334 0.270 JB24H3L3 5596400 121165190.120 JB24Chi 5780677 14683906 0.053 JB94H1L3 5605512 12073719 0.314JB94H2L1 5565211 12085615 0.214 JB94H3L3 3402301 12844937 0.456 JB94Chi4653071 12168782 0.257

Example 11 Reverse Effect of Humanized Antibody on AMP-Mediated HumanCD4+T Cell Proliferation Inhibition

This method confirmed the ability of humanized CD73 antibody to releaseAMP-mediated CD4+T cell proliferation inhibition in vitro; at the sametime, the blockade ability of humanized antibodies to CD4+T cell CD73enzyme activity was detected by CellTiter-Glo (Promega) reagent; and theIFN-γ levels in cell culture supernatant were detected by ELISA.

The results of the activities of humanized antibody of JB24Chi andJB94Chi were shown in FIG. 13. Among them, the activities of JB94H1L3,94H1L3 hIgG2 and JB94H3L3 were about 10⁻¹¹˜10⁻¹²M (Table 13).

TABLE 13 The ability of humanized antibodies reversing AMP-mediatedhuman CD4 + T cell proliferation inhibition Ab Bottom Top EC₅₀(nM)JB24H2L1 22.61 81.28 0.032 JB24H3L2 6.097 86.32 0.022 JB24H3L3 16.4585.72 0.068 JB24Chi 25.18 81.45 0.003 JB94H1L3 29.9 81.79 0.002 94H1L30.558 103.5 0.012 hIgG2 JB94H2L1 20.33 81.28 0.032 JB94H3L3 24.47 82.010.003 JB94Chi 22.86 83.57 0.003

ELISA detection kit (Dakewe) was used to detect cell supernatant IFN-γlevel. As shown in FIG. 14, except for JB24H3L3, each humanized antibodystimulated T cells to secrete IFN-γ with a EC₅₀ of 10⁻¹¹˜10⁻¹²M (Table14).

TABLE 14 The ability of humanized Cd73 antibodies reversing the releaseof IFN-γ from CD4 + T cells Ab Bottom Top EC₅₀(nM) JB24H2L1 380.5 8750.002 JB24H3L2 164.6 1527 0.015 JB24H3L3 387.6 2249 0.345 JB24Chi 474.11897 0.006 JB94H1L3 391.1 1165 0.001 94H1L3 267.7 4791 0.001 hIgG2JB94H2L1 380.4 875 0.002 JB94H3L3 245.9 1419 0.003 JB94Chi 267.4 15500.004

Example 12 Inhibition of 5′Exonuclease Activity in A375 Xenograft Modelby Humanized Antibody

B-NDG mice (Biocytogen) were subcutaneously inoculated with A375 cells,and 3 days later, the antibodies JB94H1L3, 94H3L3 hIgG1 (SEQ ID NOS: 91and 85) or PBS were injected intraperitoneally (i.p.) 2 times/week, anddosing for two weeks, tumors were harvested at 1 d, 2 d, 3 d, and 7 dafter administration. Place fresh tissue in a small cup filled withliquid nitrogen to freeze the tissue quickly, embed it in OCT, cut into5-6 μm thickness and dry at room temperature. The tumor section is fixedwith 4° C. pre-cooled acetone for 20 min. Store at −80° C. after drying.Take the tumor section out of the refrigerator at −80° C., equilibrateat room temperature for 5-10 min, put it into 50 mM Tris-maleic acidbuffer (PH7.4, containing 2 mM CaCl₂ and 0.25M sucrose), pre-incubate atroom temperature for 1 h, remove the pre-incubation buffer after 1 h,and again add 50 mM Tris-maleic acid buffer (PH7.4, containing 5 mMMnCl₂, 2 mM lead nitrate, 2.5% dextran T200, 2.5 mM levamisole and 400μM AMP), incubate at 37° C. for 1.5 h; rinse three times with water,incubate the sections with 1% (NH₄)₂S for 1 min at room temperature, andthen quickly rinse three times with water; the sections werecounterstained with hematoxylin and eosin, dehydrated, xylenetransparent, and photographed under a microscope. Brown indicates thepresence of activated CD73, and deletion of brown indicates that theantibody blocks the enzyme activity of CD73. Results are shown in FIG.15A and FIG. 15B.

The above results show that both JB94H1L3 and 94H3L3 hIgG1 cansignificantly reduce the 5′exonuclease activity of tumors in xenograftanimal models, and are related to the administration time. On the 3rdday after discontinuation, the CD73 enzyme activity in the JB94H1L3treatment group was reduced to the lowest point, and then slightlyincreased on the 7th day. The CD73 enzyme activity in the 94H3L3 hIgG1treatment group gradually increased from the first day afteradministration. In summary, both antibodies showed good effect oninhibiting CD73 activity.

Example 13 Tumor Suppressive Effect of Humanized Antibodies on A375Human Melanoma Xenograft Model

A375 human melanoma cells were inoculated subcutaneously in the righthypochondrium of NCG mice, and PBMC were resuspended in PBS and theninoculated in the mice. When the tumors grew to 50-100 mm³,administering in two groups, giving JB94H1L3 (10 mg/kg) or solventcontrol. The tumor volume was measured twice a week using verniercalipers to measure the long and short diameters of the tumor, and thevolume calculation formula is: volume=0.5 along diameter×shortdiameter². As shown in FIG. 16, JB94H1L3 exhibited significant tumorgrowth inhibition effect.

These results were obtained using the following materials and methods:

Antibody Preparation:

Preparation of hybridoma antibodies: Thawed hybridoma cells werecultured for 7-10 days in 10% FBS-containing DMEM medium, and the cellculture supernatant was collected, centrifuged at 1000 rpm for 10minutes, then the supernatants was purified with Protein G HP SpinTrap(GE Healthcare). Quantification was performed using an UV5nano(millipore) spectrophotometer.

Preparation of recombinant antibody: ExpiCHO cells (Thermo FisherScientific) with a viability of 99% were adjust to a density of 6×10⁶cells/ml. The cells were centrifuged at 1500 rpm for 5 min andresuspended. Prepare the expression vector diluent, ExpiFectamine CHOreagent diluent and DNA-ExpiFectamine CHO complex according to theinstructions, and transfer the DNA-ExpiFectamine complex to the cellculture solution. Incubate for 8 days at 37° C. in an 8% CO₂ shaker.Centrifuge at 11,000 rpm for 10 min to collect the cell supernatant.Antibodies were purified using an NGC chromatography system (Bio-Rad)and rProtein G Beads 4FF pre-assembled columns. Quantification wasperformed using an UV5nano (millipore) spectrophotometer.

Assay 1: Indirect ELISA

1 ug/ml of recombinant CD73 protein was coated on ELISA plates (Coning),incubate overnight at 4° C. The next day, wash 5 times in PBS andblocked with 200 ul/well 2% skimmed milk, a certain dose range of CD73antibodies were incubated for 1 h at room temperature. Wash 5 times withPBST washing buffer (PBS, 0.05% Tween 20), add HRP-conjugated goatanti-mouse IgG (H+L) (Promega) and incubate at room temperature for 1 hto detect bound anti-CD73 antibodies; wash the plate 5 times again andadd TMB (Life Technologies) for color development for 5 to 10 min;finally the reaction was stopped by adding 1N HCl, and the OD value wasmeasured at 450 nm. GraphPad Prism software was used to generate datagraphs and statistical affinity data.

Assay 2: Flow Cytometry

Add 100 μL 2×10⁵ cells/well expressing huCD73 (293T/17-huCD73), cynoCD73(293T/17-cynoCD73) or moCD73 (293T/17-moCD73) cells to a 96-wellU-shaped cell culture plate. The samples to be tested were incubated for1 h at 4° C. after gradient dilution. After washing, 100 μL ofAPC-labeled secondary antibody was added to each well and incubated for30 min. After washing, detecting on machine and the fluorescence valuewas read. GraphPad Prism software was used to generate data graphs andstatistical affinity data.

Construction of recombinant host cells 293T/17-huCD73, 293T/17-cynoD73,293T/17-moD73: use the PLVX-EF1a virus vector having huCD73, cynoCD73and moCD73 genes and puromycin resistance to transfect 293T/17 cellswith TransIT®-293 transfection reagent, after about 2 days, replace thecomplete medium containing puromycin to cultivate, and after the cellsare expanded and cultivated, a single cloned cell is selected using alimited gradient dilution method, and PE-labeled anti-huCD73, cynoCD73and moCD73 antibodies were used to detect cell surface antigens aftertransfection. Then the cells are expanded and cultivated for enzymeactivity assay and flow cytometry.

Assay 3: Soluble CD73 Enzyme Activity Blocking Assay

The blocking function of enzyme activity by antibodies was determinedbased on the soluble CD73 recombinant protein. In the presence of 500ng/ml CD73 recombinant protein, a certain dose range of CD73 antibodywas added to a 96-well flat bottom plate and incubated at 37° C. for 1h; then AMP and ATP with a final concentration of 180 uM and 25 uM wasadded to each well. Incubate for 30 min at 37° C.; transfer a certainvolume of cell supernatant to a blank plate, add CellTiterr-Glo(Promega) containing luciferase to the above wells at a ratio of 1:1, atroom temperature, equilibrated for 5 minutes in the dark. Finally, theEnspire microplate reader (Perkin Elmer) was used to measure theluminescence value. GraphPad Prism software was used to generate a datagraph and statistics of enzyme kinetic data.

Assess the percentage of enzyme inhibition as follows:

ATP+AMP: maxial luciferase inhibition (100%)

ATP+AMP+CD73: no luciferase inhibition (0%)

The formula of residual CD7 activity was evaluated as follows:

$\frac{\left( {{CD73} + {Ab} + {ATP} + {AMP}} \right) - \left( {{ATP} + {AMP}} \right)}{\left( {{CD73} + {ATP} + {AMP}} \right) - \left( {{ATP} + {AMP}} \right)}*100$

Assay 4: Cell CD73 Enzyme Activity Blocking Assay

The 293T/17-huCD73 cells were digested, centrifuged at 1500 rpm for 3minutes, and the supernatant was discarded. The cells were resuspendedin serum-free DMEM medium and counted with a Biorad TC20 cell counter.Adjust the cell density, spread the cells into 96-well plates at 8,000cells, 50 uL/well; add 50 uL gradient diluted antibody solution to thecorresponding wells, and incubate in a 5% CO₂ incubator for 1 hour at37° C.; then add 100 uL of AMP solution with a final concentration of180 uM and incubate in a 5% CO₂ at 37° C. for 1 hour; then centrifugethe cell plate at 1500 rpm for 3 minutes and transfer a certain volumeof culture supernatant to an opaque 96-well flat bottom plate (Costar,3912), then add 2× ATP solution to make the final reaction concentrationof 25 uM. Finally, add the corresponding volume of CellTiter Glo reagentat a ratio of 1:1 according to Promega's instructions. Afterequilibrating at room temperature for 5 minutes, read the luminescencevalue on a Perkin-Elmer Envision microplate reader and determine thecell CD73 enzyme activity by measuring the ATP level. GraphPad Prismsoftware was used to generate data graphs and statistical enzyme kineticdata.

Assay 5: Fab ZAP Endocytosis Experiment

On the first day, 2000 cells/well of MDA-MB-231 cells were plated into96-well flat bottom plates at 50 ul/well and incubated overnight at 37°C. in 5% CO₂; The next day, the antibodies were gradient diluted with 80nM Fab-ZAP reagent (Advanced Targeting Systems) to a certain dose range,and incubated for 30 min at room temperature to bind Fab-ZAP to theantibody to be tested, then the antibodies were premixed, and 50 uL ofthe mixture were added to MDA-MB-231 cells wells, after 3 daysincubation at 37° C. in 5% CO2, take the cell plate and add CTG reagent(Promega) to lyse the cells for 2 minutes, and then equilibrated theplates at room temperature for 5 minutes. Luminescences were measuredusing Enspire microplate reader (Perkin Elmer) and cell proliferationcurves were analyzed by GraphPad Prism software.

1-71. (canceled)
 72. An isolated antibody or antigen-binding fragmentthereof comprising a heavy chain variable region that comprises HCDR1,HCDR2, HCDR3; and a light chain variable region that comprises LCDR1,LCDR2, LCDR3, wherein: (a) HCDR1 comprises the amino acid sequence ofSEQ ID NO: 18; (b) HCDR2 comprises the amino acid sequence of SEQ IDNO:19; (c) HCDR3 comprises the amino acid sequence of SEQ ID NO:20; (d)LCDR1 comprises the amino acid sequence of SEQ ID NO:23; (e) LCDR2comprises the amino acid sequence of SEQ ID NO:24; and (f) LCDR3comprises the amino acid sequence of SEQ ID NO:
 25. 73. The isolatedantibody or antigen-binding fragment thereof of claim 72, whichcomprises: (i) a heavy chain variable region (VH) comprising an aminoacid sequence which has at least 85% identity to the amino acid sequenceof any one of SEQ ID NOs: 16, and 66-67, and comprises HCDR1 comprisingSEQ ID NO: 18, HCDR2 comprising SEQ ID NO: 19, and HCDR3 comprising SEQID NO: 20; and (ii) a light chain variable region (VL) comprising anamino acid sequence which has at least 85% identity to the amino acidsequence of any one of SEQ ID NO: 21, and 68-70, and comprises LCDR1comprising SEQ ID NO: 23, LCDR2 comprising SEQ ID NO: 24, and LCDR3comprising SEQ ID NO:
 25. 74. The isolated antibody or antigen-bindingfragment thereof of claim 72, which comprises: 1) a heavy chain variableregion (VH) that comprises an amino acid sequence which has at least 85%identity to the amino acid sequence of SEQ ID NO: 16 and comprises HCDR1comprising SEQ ID NO: 18, HCDR2 comprising SEQ ID NO: 19, and HCDR3comprising SEQ ID NO: 20, and a light chain variable region (VL) thatcomprises an amino acid sequence which has at least 85% identity to theamino acid sequence of SEQ ID NO:21 and comprises LCDR1 comprising SEQID NO: 23, LCDR2 comprising SEQ ID NO: 24, and LCDR3 comprising SEQ IDNO: 25; 2) a heavy chain variable region (VH) that comprises an aminoacid sequence which has at least 85% identity to the amino acid sequenceof SEQ ID NO: 66 and comprises HCDR1 comprising SEQ ID NO: 18, HCDR2comprising SEQ ID NO: 19, and HCDR3 comprising SEQ ID NO: 20, and alight chain variable region (VL) that comprises an amino acid sequencewhich has at least 85% identity to the amino acid sequence of SEQ ID NO:68 and comprises LCDR1 comprising SEQ ID NO: 23, LCDR2 comprising SEQ IDNO: 24, and LCDR3 comprising SEQ ID NO: 25; 3) a heavy chain variableregion (VH) that comprises an amino acid sequence which has at least 85%identity to the amino acid sequence of SEQ ID NO: 66 and comprises HCDR1comprising SEQ ID NO: 18, HCDR2 comprising SEQ ID NO: 19, and HCDR3comprising SEQ ID NO: 20, and a light chain variable region (VL) thatcomprises an amino acid sequence which has at least 85% identity to theamino acid sequence of SEQ ID NO: 69 and comprises LCDR1 comprising SEQID NO: 23, LCDR2 comprising SEQ ID NO: 24, and LCDR3 comprising SEQ IDNO: 25; 4) a heavy chain variable region (VH) that comprises an aminoacid sequence which has at least 85% identity to the amino acid sequenceof SEQ ID NO: 66 and comprises HCDR1 comprising SEQ ID NO: 18, HCDR2comprising SEQ ID NO: 19, and HCDR3 comprising SEQ ID NO: 20, and alight chain variable region (VL) that comprises an amino acid sequencewhich has at least 85% identity to the amino acid sequence of SEQ ID NO:70 and comprises LCDR1 comprising SEQ ID NO: 23, LCDR2 comprising SEQ IDNO: 24, and LCDR3 comprising SEQ ID NO: 25; 5) a heavy chain variableregion (VH) that comprises an amino acid sequence which has at least 85%identity to the amino acid sequence of SEQ ID NO: 67 and comprises HCDR1comprising SEQ ID NO: 18, HCDR2 comprising SEQ ID NO: 19, and HCDR3comprising SEQ ID NO: 20, and a light chain variable region (VL) thatcomprises an amino acid sequence which has at least 85% identity to theamino acid sequence of SEQ ID NO: 68 and comprises LCDR1 comprising SEQID NO: 23, LCDR2 comprising SEQ ID NO: 24, and LCDR3 comprising SEQ IDNO: 25; 6) a heavy chain variable region (VH) that comprises an aminoacid sequence which has at least 85% identity to the amino acid sequenceof SEQ ID NO: 67 and comprises HCDR1 comprising SEQ ID NO: 18, HCDR2comprising SEQ ID NO: 19, and HCDR3 comprising SEQ ID NO: 20, and alight chain variable region (VL) that comprises an amino acid sequencewhich has at least 85% identity to the amino acid sequence of SEQ ID NO:69 and comprises LCDR1 comprising SEQ ID NO: 23, LCDR2 comprising SEQ IDNO: 24, and LCDR3 comprising SEQ ID NO: 25; or 7) a heavy chain variableregion (VH) that comprises an amino acid sequence which has at least 85%identity to the amino acid sequence of SEQ ID NO: 67 and comprises HCDR1comprising SEQ ID NO: 18, HCDR2 comprising SEQ ID NO: 19, and HCDR3comprising SEQ ID NO: 20, and a light chain variable region (VL) thatcomprises an amino acid sequence which has at least 85% identity to theamino acid sequence of SEQ ID NO: 70 and comprises LCDR1 comprising SEQID NO: 23, LCDR2 comprising SEQ ID NO: 24, and LCDR3 comprising SEQ IDNO:
 25. 75. The isolated antibody or antigen-binding fragment thereof ofclaim 72, wherein the isolated antibody is an IgG.
 76. The isolatedantibody or antigen-binding fragment thereof of claim 72, wherein theisolated antibody is an IgG1, IgG2 or IgG4.
 77. The isolated antibody orantigen-binding fragment thereof of claim 72, wherein the isolatedantibody is a monoclonal antibody, a chimeric antibody, a humanizedantibody, a human engineered antibody, a human antibody, Fv, a singlechain antibody (scFv), Fab, Fab′, Fab′-SH or F(ab′)₂.
 78. The isolatedantibody or antigen-binding fragment thereof of claim 72, comprising aheavy chain and a light chain, wherein: (I) the heavy chain comprisingan amino acid sequence which has at least 85% identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 26, 27, 71and 72, and comprises HCDR1 comprising SEQ ID NO: 18, HCDR2 comprisingSEQ ID NO: 19, and HCDR3 comprising SEQ ID NO: 20; and (II) the lightchain comprising an amino acid sequence which has at least 85% identityto an amino acid sequence selected from the group consisting of SEQ IDNOs: 28, 73, 74 and 75, and comprises LCDR1 comprising SEQ ID NO: 23,LCDR2 comprising SEQ ID NO: 24, and LCDR3 comprising SEQ ID NO:
 25. 79.The isolated antibody or antigen-binding fragment thereof of claim 72,which comprises: 1) a heavy chain that comprises an amino acid sequencewhich has at least 85% identity to the amino acid sequence of SEQ ID NO:26 and comprises HCDR1 comprising SEQ ID NO: 18, HCDR2 comprising SEQ IDNO: 19, and HCDR3 comprising SEQ ID NO: 20, and a light chain thatcomprises an amino acid sequence which has at least 85% identity to theamino acid sequence of SEQ ID NO: 28 and comprises LCDR1 comprising SEQID NO: 23, LCDR2 comprising SEQ ID NO: 24, and LCDR3 comprising SEQ IDNO: 25; 2) a heavy chain that comprises an amino acid sequence which hasat least 85% identity to the amino acid sequence of SEQ ID NO: 27 andcomprises HCDR1 comprising SEQ ID NO: 18, HCDR2 comprising SEQ ID NO:19, and HCDR3 comprising SEQ ID NO: 20, and a light chain that comprisesan amino acid sequence which has at least 85% identity to the amino acidsequence of SEQ ID NO: 28 and comprises LCDR1 comprising SEQ ID NO: 23,LCDR2 comprising SEQ ID NO: 24, and LCDR3 comprising SEQ ID NO: 25; 3) aheavy chain that comprises an amino acid sequence which has at least 85%identity to the amino acid sequence of SEQ ID NO: 71 and comprises HCDR1comprising SEQ ID NO: 18, HCDR2 comprising SEQ ID NO: 19, and HCDR3comprising SEQ ID NO: 20, and a light chain that comprises an amino acidsequence which has at least 85% identity to the amino acid sequence ofSEQ ID NO: 73 and comprises LCDR1 comprising SEQ ID NO: 23, LCDR2comprising SEQ ID NO: 24, and LCDR3 comprising SEQ ID NO: 25; 4) a heavychain that comprises an amino acid sequence which has at least 85%identity to the amino acid sequence of SEQ ID NO: 71 and comprises HCDR1comprising SEQ ID NO: 18, HCDR2 comprising SEQ ID NO: 19, and HCDR3comprising SEQ ID NO: 20, and a light chain that comprises an amino acidsequence which has at least 85% identity to the amino acid sequence ofSEQ ID NO: 74 and comprises LCDR1 comprising SEQ ID NO: 23, LCDR2comprising SEQ ID NO: 24, and LCDR3 comprising SEQ ID NO: 25; 5) a heavychain that comprises an amino acid sequence which has at least 85%identity to the amino acid sequence of SEQ ID NO: 71 and comprises HCDR1comprising SEQ ID NO: 18, HCDR2 comprising SEQ ID NO: 19, and HCDR3comprising SEQ ID NO: 20, and a light chain that comprises an amino acidsequence which has at least 85% identity to the amino acid sequence ofSEQ ID NO: 75 and comprises LCDR1 comprising SEQ ID NO: 23, LCDR2comprising SEQ ID NO: 24, and LCDR3 comprising SEQ ID NO: 25; 6) a heavychain that comprises an amino acid sequence which has at least 85%identity to the amino acid sequence of SEQ ID NO: 72 and comprises HCDR1comprising SEQ ID NO: 18, HCDR2 comprising SEQ ID NO: 19, and HCDR3comprising SEQ ID NO: 20, and a light chain that comprises an amino acidsequence which has at least 85% identity to the amino acid sequence ofSEQ ID NO: 73 and comprises LCDR1 comprising SEQ ID NO: 23, LCDR2comprising SEQ ID NO: 24, and LCDR3 comprising SEQ ID NO: 25; 7) a heavychain that comprises an amino acid sequence which has at least 85%identity to the amino acid sequence of SEQ ID NO: 72 and comprises HCDR1comprising SEQ ID NO: 18, HCDR2 comprising SEQ ID NO: 19, and HCDR3comprising SEQ ID NO: 20, and a light chain that comprises an amino acidsequence which has at least 85% identity to the amino acid sequence ofSEQ ID NO: 74 and comprises LCDR1 comprising SEQ ID NO: 23, LCDR2comprising SEQ ID NO: 24, and LCDR3 comprising SEQ ID NO: 25; or 8) aheavy chain that comprises an amino acid sequence which has at least 85%identity to the amino acid sequence of SEQ ID NO: 72 and comprises HCDR1comprising SEQ ID NO: 18, HCDR2 comprising SEQ ID NO: 19, and HCDR3comprising SEQ ID NO: 20, and a light chain that comprises an amino acidsequence which has at least 85% identity to the amino acid sequence ofSEQ ID NO: 75 and comprises LCDR1 comprising SEQ ID NO: 23, LCDR2comprising SEQ ID NO: 24, and LCDR3 comprising SEQ ID NO:
 25. 80. Theisolated antibody or antigen-binding fragment thereof of claim 79,wherein the antibody comprises: 1) a heavy chain consisting of SEQ IDNO: 26 and a light chain consisting of SEQ ID NO: 28; 2) a heavy chainconsisting of SEQ ID NO: 27 and a light chain consisting of SEQ ID NO:28; 3) a heavy chain consisting of SEQ ID NO: 71 and a light chainconsisting of SEQ ID NO: 73; 4) a heavy chain consisting of SEQ ID NO:71 and a light chain consisting of SEQ ID NO: 74; 5) a heavy chainconsisting of SEQ ID NO: 71 and a light chain consisting of SEQ ID NO:75; 6) a heavy chain consisting of SEQ ID NO: 72 and a light chainconsisting of SEQ ID NO: 73; 7) a heavy chain consisting of SEQ ID NO:72 and a light chain consisting of SEQ ID NO: 74; or 8) a heavy chainconsisting of SEQ ID NO: 72 and a light chain consisting of SEQ ID NO:75.
 81. The isolated antibody or antigen-binding fragment thereof ofclaim 72, which is an antagonist of CD73 or an antagonist of5′-nucleotidase of CD73.
 82. The isolated antibody or antigen-bindingfragment thereof of claim 81, wherein the CD73 is human CD73.
 83. Amethod of decreasing adenosine levels in a subject with tumor, improvinga T cell response in a subject with tumor, stimulating an immuneresponse in a subject, or inhibiting the growth of tumor cells in asubject, said method comprises administering a therapeutically effectiveamount of the isolated antibody or antigen-binding fragment thereof ofclaim 72 to the subject.
 84. The method of claim 83, wherein the subjectis a subject with tumor.
 85. The method of claim 83, wherein the subjecthas a tumor cell expressing CD73 and/or a tumor microenvironmentcontaining CD73.
 86. A pharmaceutical composition comprising theisolated antibody or antigen-binding fragment thereof of claim 72, and apharmaceutically acceptable excipient.
 87. A method of treating tumorcomprising administering a therapeutically effective amount of theisolated antibody or antigen-binding fragment thereof of claim 72 to asubject in need thereof.
 88. The method of claim 87, wherein the tumoris a solid tumor or hematological tumor.
 89. The method of claim 87,wherein the tumor is selected from bladder cancer, breast cancer,cervical cancer, ovarian cancer, prostate cancer, testicular cancer,esophageal cancer, gastrointestinal cancer, pancreatic cancer,colorectal cancer, colon cancer, renal cancer, head and neck cancer,lung cancer (small cell lung cancer or non-small cell lung cancer),stomach cancer, bone cancer, liver cancer, thyroid cancer, skin cancer,central nervous system tumor, lymphoma, leukemia, myeloma, sarcoma, andvirus-associated cancer.